Methods of attaching a molecule-of-interest to a microtube

ABSTRACT

A method of attaching a molecule-of-interest to a microtube, by co-electrospinning two polymeric solutions through co-axial capillaries, wherein a first polymeric solution of the two polymeric solutions is for forming a shell of the microtube and a second polymeric solution of the two polymeric solutions is for forming a coat over an internal surface of the shell, the first polymeric solution is selected solidifying faster than the second polymeric solution and a solvent of the second polymeric solution is selected incapable of dissolving the first polymeric solution and the second polymeric solution comprises the molecule-of-interest, thereby attaching the molecule-of-interest to the microtube. An electrospun microtube comprising an electrospun shell, an electrospun coat over an internal surface of the shell and a molecule-of-interest attached to the microtube.

RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Nos.61/064,210, 61/064,206 and 61/064,204 filed on Feb. 21, 2008.

The teachings of PCT/IB2007/054001 are incorporated herein by reference.

The contents of all of the above documents are incorporated by referenceas if fully set forth herein.

FIELD AND BACKGROUND OF THE INVENTION

The invention, in some embodiments thereof, relates to a method ofattaching a molecule-of-interest to a microtube and, more particularly,but not exclusively, to electrospun microtubes including themolecule-of-interest attached thereto.

In nature there is an enormous variety of enzymes that catalyzereactions, some of which have industrial use. These includeoxidoreductases, transferases, hydrolases, lyases, isomerases, andligases. Immobilization of enzymes on solid substrates sometimes offersadvantages over the use of a free enzyme. For example, immobilizationcan stabilize enzymes, enable better control of enzymatic reactions,allow the reuse of the enzyme and prevent enzyme loss with time. Thematerial bearing the immobilized enzyme has a significant role inevoking these advantages both from architectural and chemical points ofview.

Nanofibers and polymeric nanofibers in particular can be produced by anelectrospinning process (Reneker D H., et al., 2006; Ramakrishna S., etal., 2005; Li D., et al., 2004; PCT WO 2006/106506 to the presentinventors). Electrospun polymeric nanofibers have been widely used inbiological applications such as scaffolds, carriers for biologicallyactive molecules like proteins and enzymes (Xie J., et al., 2003; ZhangY Z., et al., 2006; Jiang H., et al., 2006; and Patel A C., et al.,2006) and encapsulation of viruses and bacteria (Salalha W., et al.,2006).

Several approaches can be used to entrap or attach enzymes toelectrospun fibers. One approach is to immobilize the enzyme on theouter surface of the nanofibers by either covalently attaching thedesired enzyme to the functional groups of the polymer surface (Ye P.,et al., 2006; Jia H., et al., 2002; Kim T G., et al., 2006) orphysically absorbing the enzyme to the surface (Huang X J., et al.,2006). The second approach, which results in encapsulation of enzymes,is based on mixing the enzyme with the polymer solution prior to theelectrospinning process (Xie J. and Hsieh Y-L, 2003). However,encapsulation is often associated with leaching of the enzymes, e.g.,via fiber dissolution and burst releases (Zhang Y Z., et al., 2006),especially, when the host polymer is a water soluble polymer such aspoly(vinyl alcohol) (PVA) or dextran. To prevent immediate dissolutionof the fibers in a physiological environment (e.g., blood) and thesubsequent enzyme leaching, the electrospun fibers can be crosslinked bychemical or physical agents such as glutaraldehyde or UV irradiation.Alternatively, Zeng J, et al. (2005) suggested that PVA fibers can becoated with water insoluble polymers using a chemical vapor deposition(CVD). However, the organic solvents of the water insoluble polymers areharmful to biological material and can lead to loss of enzymaticactivity. To overcome this problem, Herricks et al. (2005) suggested touse surfactant-stabilized enzymes in an organic solution of polystyrene(PS) as a spinning solution. In this way the electrospun nanofibers areinsoluble in water and the enzymatic activity is retained due tosurfactant stabilization (Herricks T E., et al., 2005).

Sun and co-workers (Sun Z, et al., 2003) describe the production ofcore-shell nanofibers (i.e., filled fibers) by co-electrospinning of twopolymeric solutions using a two co-axial capillaries spinneret. USpatent application No. 20060119015 to Wehrspohn R., et al. describes theproduction of hollow fibers by introducing a liquid containing a polymerto a porous template material, and removal of the template followingpolymer solidification. PCT/IB2007/054001 to the present inventors(which is fully incorporated herein by reference) discloses methods ofproducing electrospun microtubes (i.e., hollow fibers) which can befurther filled with liquids and be used as microfluidics.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a method of attaching a molecule-of-interest to amicrotube, the method comprising: co-electrospinning two polymericsolutions through co-axial capillaries, wherein a first polymericsolution of the two polymeric solutions is for forming a shell of themicrotube and a second polymeric solution of the two polymeric solutionsis for forming a coat over an internal surface of the shell, the firstpolymeric solution is selected solidifying faster than the secondpolymeric solution and a solvent of the second polymeric solution isselected incapable of dissolving the first polymeric solution andwherein the second polymeric solution comprises themolecule-of-interest, thereby attaching the molecule-of-interest to themicrotube.

According to an aspect of some embodiments of the present inventionthere is provided a microtube comprising an electrospun shell, anelectrospun coat over an internal surface of the shell and amolecule-of-interest attached to the microtube.

According to an aspect of some embodiments of the present inventionthere is provided a method of processing a substrate-of-interest,comprising contacting the substrate-of-interest with the microtube ofthe invention, wherein the molecule-of-interest is capable of processingthe substrate, thereby processing the substrate-of-interest.

According to an aspect of some embodiments of the present inventionthere is provided a method of depleting a molecule from a solution,comprising contacting the solution with the microtube of the invention,wherein the member of the affinity pair is selected capable of bindingthe molecule, thereby depleting the molecule from the solution.

According to an aspect of some embodiments of the present inventionthere is provided a method of isolating a molecule from a solution,comprising: (a) contacting the solution with the microtube of theinvention under conditions which allow binding of the molecule to themicrotube via the member of the affinity pair which is selected capableof binding the molecule, and; (b) eluting the molecule from themicrotube; thereby isolating the molecule from the solution.

According to an aspect of some embodiments of the present inventionthere is provided a method of detecting a presence of a molecule in asample, comprising: (a) contacting the sample with the microtube of theinvention, wherein the member of the affinity pair is selected capableof binding the molecule, and; (b) detecting binding of the molecule bythe member of the affinity pair; thereby detecting the presence of amolecule in the sample.

According to an aspect of some embodiments of the present inventionthere is provided a method of releasing a molecule-of-interest to cellsof a subject in need thereof, comprising implanting in the subject themicrotube of the invention, to thereby release the molecule-of-interestto cells of the subject.

According to some embodiments of the invention, the electrospun shell isformed of a first polymeric solution and the electrospun coat is formedof a second polymeric solution.

According to some embodiments of the invention, the first polymericsolution solidifies faster than the second polymeric solution.

According to some embodiments of the invention, a solvent of the secondpolymeric solution is incapable of dissolving the first polymericsolution.

According to some embodiments of the invention, the electrospun shellcomprises a polymer selected from the group consisting of poly(e-caprolactone) (PCL), polyamide, poly(siloxane), poly(silicone),poly(ethylene), poly(vinyl pyrrolidone), poly(2-hydroxyethylmethacrylate), poly(N-vinyl pyrrolidone), poly(methylmethacrylate), poly(vinyl alcohol), poly(acrylic acid), poly(vinylacetate), polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethyleneglycol), poly(methacrylic acid), polylactide, polyglycolide,poly(lactide-coglycolide), polyanhydride, polyorthoester,poly(carbonate), poly(acrylo nitrile), poly(ethylene oxide),polyaniline, polyvinyl carbazole, polystyrene, poly(vinyl phenol),polyhydroxy acid, poly(caprolactone), polyanhydride,polyhydroxyalkanoate, polyurethane, collagen, albumin, alginate,chitosan, starch, hyaluronic acid, and whereas the electrospun coatcomprises a polymer selected from the group consisting of poly(acrylicacid), poly(vinyl acetate), polyacrylamide, poly(ethylene-co-vinylacetate), poly(ethylene glycol), poly(methacrylic acid), polylactidepolyglycolide, poly(lactide-coglycolide), polyanhydride, polyorthoester,poly(carbonate), poly(ethylene oxide), polyaniline, polyvinyl carbazole,polystyrene, poly(vinyl phenol), polyhydroxy acid, alginate, starch,hyaluronic acid.

According to some embodiments of the invention, a solvent of the firstpolymeric solution evaporates faster than a solvent of the secondpolymeric solution.

According to some embodiments of the invention, the electrospinning iseffected using a rotating collector.

According to some embodiments of the invention, a solvent of the secondpolymeric solution is capable of evaporating through the internalsurface of the shell.

According to some embodiments of the invention, the second polymericsolution is capable of wetting the internal surface of the shell.

According to some embodiments of the invention, a thickness of the shellis from about 100 nm to about 20 micrometer.

According to some embodiments of the invention, an internal diameter ofthe microtube is from about 50 nm to about 20 micrometer.

According to some embodiments of the invention, the first and the secondpolymeric solutions are selected from the group consisting of: 10% poly(e-caprolactone) (PCL) in chloroform (CHCl₃) and dimethylforamide (DMF)(80:20 by weight) as the first polymeric solution and 4% poly(ethyleneoxide) (PEO) in water (H₂O) and ethanol (60:40 by weight) as the secondpolymeric solution, 10% PCL in CHCl₃ and DMF (80:20 by weight) as thefirst polymeric solution and 6% PEO in H₂O and ethanol (60:40 by weight)as the second polymeric solution, 9% PCL in CHCl₃ and DMF (90:10 byweight) as the first polymeric solution and 7% PEO in H₂O as the secondpolymeric solution, 10% PCL in CHCl₃ and DMF (80:20 by weight) as thefirst polymeric solution and 9% poly(vinyl alcohol) (PVA) in water andethanol (50:50 by weight) as the second polymeric solution, and 10% PCLin CHCl₃ and DMF (90:10 by weight) as the first polymeric solution and4% (w/w) PEO in ethanol:H₂O (26:74 by weight) as a second polymericsolution.

According to some embodiments of the invention, the microtube is filledwith a liquid.

According to some embodiments of the invention, the first and the secondpolymeric solutions are biocompatible.

According to some embodiments of the invention, the molecule-of-interestis attached to the coat over the internal surface of the shell.

According to some embodiments of the invention, the molecule-of-interestis attached to the shell of the microtube.

According to some embodiments of the invention, the molecule-of-interestcomprises a polypeptide, a polynucleotide, a carbohydrate, a smallmolecule, or any combination thereof.

According to some embodiments of the invention, the molecule-of-interestcomprises a member of an affinity pair.

According to some embodiments of the invention, the polypeptide is anenzyme.

According to some embodiments of the invention, the enzyme is alkalinephosphatase (SEQ ID NO: 1 or 8) or beta-galactosidase (SEQ ID NO:2 or9).

According to some embodiments of the invention, the first polymericsolution comprises polyethylene glycol (PEG).

According to some embodiments of the invention, the shell comprisespores.

According to some embodiments of the invention, the shell preventsdiffusion of the molecule-of-interest therethrough.

According to some embodiments of the invention, thesubstrate-of-interest comprises incorporating the substrate-of-interestin a synthesis reaction catalyzed by the molecule-of-interest.

According to some embodiments of the invention, thesubstrate-of-interest comprises incorporating the substrate-of-interestin a catabolism reaction catalyzed by the molecule-of-interest.

According to some embodiments of the invention, the method furthercomprising collecting the solution following the contacting.

According to some embodiments of the invention, the solution comprisesblood.

According to some embodiments of the invention, the affinity pair isselected from the group consisting of an enzyme and a substrate, ahormone and a receptor, an antibody and an antigen, a polypeptide and apolynucleotide, a polynucleotide and a cognate polynucleotide, apolypeptide and a metal ion, a polypeptide and a carbohydrate.

According to some embodiments of the invention, a therapeuticallyeffective amount of the molecule-of-interest is capable of treating apathology in the subject.

According to some embodiments of the invention, the molecule-of-interestcomprises a polypeptide, and whereas a therapeutically effective amountof the polypeptide is capable of treating a pathology in the subject.

According to some embodiments of the invention, the pathology isselected from the group consisting of a metabolic disorder, an endocrinedisease, an autoimmune disease, and cancer.

According to some embodiments of the invention, the polypeptide isselected from the group consisting of insulin (SEQ ID NO:6),phenylalanine hydroxylase (PAH) (SEQ ID NO:3), dystrophin (SEQ ID NO:4),beta-glucosidase (GBA) (SEQ ID NO:5), and ceruloplasmin ferroxidase (CP)(SEQ ID NO:7).

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-D are images depicting high resolution scanning electronmicroscope (HRSEM) micrographs of (a) Type 1 fibers with alkalinephosphatase (AP); (b) Type 2 fibers with AP; (c) Type 1 fibers withbeta-galactosidase (β-GAL); and (d) Type 2 fibers with β-GAL;

FIGS. 2A-C are graphs (FIGS. 2a-b ) and a picture (FIG. 2C) depictingthe progress of the AP reactions with time for enzymes attached to(e.g., encapsulated within) the electrospun fibers and the free enzymein the solution forming the coat over the internal surface of the shell(also referred to herein as a core solution) prior to theelectrospinning. FIG. 2A—the progress of the alkaline phosphatasereaction over 2500 minutes; FIG. 2B—inset, the progress of the reactionthrough over the first 300 minutes; Note that the enzymatic reaction ofthe enzyme encapsulated within type 2 electrospun microtubes is fasterthan that of enzyme encapsulated within type 1 electrospun microtubes.FIG. 2C—a photograph of a piece of mat (Type 1) immersed in the assaysolution. The presence of the yellow reaction product, p-nitrophenol, isapparent; the reaction substrate was para-nitrophenyl phosphate;

FIG. 3 is a histogram depicting the relative activity of AP enzyme fordifferent types of fibers (type 1 and type 2) and in dwelling buffers.Mat=the electrospun fibers (microtubes) with the attached (encapsulated)enzymes; rinsing buffer=the buffer used to only rinse the fibers,without any additional incubation time; 24 hrs. buffer=the bufferfollowing incubation of the fibers therein for 24 hours; 72 hrs.buffer=the buffer following incubation of the fibers therein for 72hours; core solution=the enzyme in the core solution prior to theelectrospinning process.

FIGS. 4A-B are graphs depicting the progress of the β-GAL reactions withtime for the two types of electrospun fibers and the free enzyme in thecore solution. The substrate was ortho-nitrophenyl galactoside. FIG.4A—the progress of the β-GAL reactions over 5000 minutes as measured bythe amount of ortho-nitrophenol generated; FIG. 4B—inset of the graph ofFIG. 4a , the progress of the β-GAL reactions over the first 50 minutes;

FIG. 5 is a graph depicting the β-GAL reaction versus time for the matand buffers for Type 2 fibers;

FIG. 6 is a histogram depicting the relative activity of the β-GAL andAP for different types of fibers;

FIGS. 7A-B are fluorescence microscope micrographs depicting Type 1fibers with AP (FIG. 7A) and β-GAL (FIG. 7B). Size bars: 100 μm (FIG.7A) and 50 μm (FIG. 7B).

FIG. 8 is a schematic illustration depicting the desorption process ofthe molecule-of-interest from the microtube of the invention. Themolecule-of-interest (e.g., a protein, an enzyme) is attached to thecoat over the internal surface of the shell. Following contacting themicrotube with a solution, the solution enters the microtube via thepores (an exemplary pore is marked by arrow No. 3) by a capillary rise(see arrow No. 1) and gradually wets and fills the microtube innervolume. The desorption of the molecule-of-interest from the internalsurface of the microtube shell (which depends mainly on the rate of therelease of the molecule-of-interest from the polymer) is shown by arrowNo. 2.

FIG. 9 depicts a multi-step enzymatic reaction performed usingencapsulated molecules-of-interest (enzymes 1-4). Enzyme 1 (enz1)catalyzes the conversion of compound A to B; Enzyme 2 (enz2) catalyzesthe conversion of compound B to C; Enzyme 3 (enz3) catalyzes theconversion of compound C to D; Enzyme 4 (enz4) catalyzes the conversionof compound D to E.

FIG. 10 is a schematic presentation depicting Atrazine degradation bythe isolated Pseudomonas ADP enzymes: AtzA (atrazine chlorohydrolase,e.g., GenBank Accession No. NP_862474), AtzB (hydroxyatrazine hydrolase,e.g., GenBank Accession No. NP_862481), AtzC (N-isopropylammelideisopropylamino hydrolase, e.g., GenBank Accession No. NP_862508), AtzD(cyanuric acid amidohydrolase, e.g., GenBank Accession No NP_862537),AtzE (biuret hydrolase, e.g., GenBank Accession No. NP_862538) and AtzF(allophanate hydrolase, e.g., GenBank Accession No. AAK50333) which areattached to the microtube of the invention.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to methodsof attaching a molecule-of-interest to a microtube and, moreparticularly, but not exclusively, to electrospun microtubes whichinclude a molecule-of-interest attached thereto which can be used invarious therapeutic, diagnostic, purification and synthesisapplications.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

While reducing the invention to practice, the present inventors haveuncovered that a molecule-of-interest can be attached to electrospunmicrotubes. Thus, as is shown in FIGS. 1a-b and described in Example 1of the Examples section which follows, two types of electrospunmicrotubes containing active molecules-of-interest (e.g., enzymes suchas alkaline phosphatase or beta-galactosidase) were formed: Type 1microtubes which exhibit a non-porous shell, and Type 2 microtubes whichexhibit a porous shell due to the presence of PEG in the polymericsolution forming the shell. The enzymatic activity contained within themicrotubes was at the same order of magnitude as that of the polymericsolution prior to the electrospinning process (FIGS. 2a-b , 3, 4 a-b and6; Examples 1-3 of the Examples section which follows) indicating thatsurprisingly the process of production did not compromise thefunctionality of the delicate protein material incorporated into thetube. In addition, as is shown in FIG. 3 and described in Example 2 ofthe Examples section which follows, both the porous and non-porousmicrotubes were capable of releasing enzymes attached thereto. Moreover,during the electrospinning process, some of the alkaline phosphataseenzyme migrated to the outer surface of the microtube shell and wasreleased therefrom into the aqueous environment (FIG. 3, Example 2),while the β-GAL enzyme remained within the internal surface of the shell(FIG. 5, Example 3). In addition, as is further described in Example 3of the Examples section which follows, the activity of the enzymesattached to the internal surface of the shell was increased in thepresence of a porous shell which enabled the passage of substratestherethrough (FIGS. 4a-b ). These results support the use of themicrotubes of the invention as micro-reactors (e.g., bioreactors) forvarious synthesis, hydrolysis, isolation and purification reactions.

According to an aspect of the invention there is provided a method ofattaching a molecule-of-interest to a microtube. The method is effectedby co-electrospinning two polymeric solutions through co-axialcapillaries, wherein a first polymeric solution of the two polymericsolutions is for forming a shell of the microtube and a second polymericsolution of the two polymeric solutions is for forming a coat over aninternal surface of the shell, the first polymeric solution is selectedsolidifying faster than the second polymeric solution and a solvent ofthe second polymeric solution is selected incapable of dissolving thefirst polymeric solution and wherein the second polymeric solutioncomprises the molecule-of-interest, thereby attaching themolecule-of-interest to the microtube.

As used herein the term “microtube” refers to a hollow tube having aninner diameter of e.g., about 200 nm to about 50 μm and an outerdiameter of e.g., about 0.5 μm to about 100 μm.

According to some embodiments of the invention the thickness of themicrotube shell can vary from a few nanometers to several micrometers,such as from about 100 nm to about 20 μm, e.g., from about 200 nm toabout 10 μm, from about 100 nm to about 5 μm, from about 100 nm to about1 μm, e.g., about 500 nm.

According to some embodiments of the invention the internal diameter ofthe microtube shell can vary from a few nanometers to severalmicrometers, such as from about 50 nm to about 50 μm, e.g., from about100 nm to about 20 μm, from about 200 nm to about 10 μm, from about 500nm to about 5 μm, from about 1 μm to about 5 μm, e.g., about 3 μm.

According to some embodiments of the invention, the microtube may have alength which is from about 0.1 millimeter (mm) to about 20 centimeter(cm), e.g., from about 1-20 cm, e.g., from about 5-10 cm.

As used herein the term “attaching” refers to the binding of themolecule-of-interest to the polymer(s) comprised in the microtube of theinvention via covalent or non-covalent binding (e.g., via anelectrostatic bond, a hydrogen bond, a van-Der Waals interaction) so asto obtain an absorbed, embedded or immobilized molecule-of-interest tothe microtube of the invention.

According to some embodiments of the invention, the length (L) of themicrotube can be several orders of magnitude higher (e.g., 10 times, 100times, 1000 times, 10,000 times) than the microtube's diameter (D).Accordingly, a molecule-of-interest which is attached to such amicrotube is referred to as being entrapped or encapsulated within themicrotube.

According to some embodiments of the invention, covalent attachment ofthe molecule-of-interest can be via functional groups such as SH groups,amino groups, carboxyl groups which are added to the polymer(s) formingthe microtube.

As used herein the phrase “co-electrospinning” refers to a process inwhich at least two polymeric solutions are electrospun from co-axialcapillaries (i.e., at least two capillary dispensers wherein onecapillary is placed within the other capillary while sharing a co-axialorientation) forming the spinneret within an electrostatic field in adirection of a collector. The capillary can be, for example, a syringewith a metal needle or a bath provided with one or more capillaryapertures from which the polymeric solution can be extruded, e.g., underthe action of hydrostatic pressure, mechanical pressure, air pressureand/or high voltage.

The collector serves for collecting the electrospun element (e.g., theelectrospun microtube) thereupon. Such a collector can be a rotatingcollector or a static (non rotating) collector. When a rotatingcollector is used, such a collector may have a cylindrical shape (e.g.,a drum), however, the rotating collector can be also of a planargeometry (e.g., an horizontal disk). The spinneret is typicallyconnected to a source of high voltage, such as of positive polarity,while the collector is grounded, thus forming an electrostatic fieldbetween the dispensing capillary (dispenser) and the collector.Alternatively, the spinneret can be grounded while the collector isconnected to a source of high voltage, such as with negative polarity.As will be appreciated by one ordinarily skilled in the art, any of theabove configurations establishes motion of a positively charged jet fromthe spinneret to the collector. Reverse polarity for establishingmotions of a negatively charged jet from the spinneret to the collectorare also contemplated.

For electrospinning, the first polymeric solution is injected into theouter capillary of the co-axial capillaries while the second polymericsolution is injected into the inner capillary of the co-axialcapillaries. In order to form a microtube (i.e., a hollow structure, asmentioned above), the first polymeric solution (which is for forming theshell of the microtube) solidifies faster than the second polymericsolution (also referred herein as a core polymeric solution, and is forforming a coat over the internal surface of the shell). In addition, theformation of a microtube also requires that the solvent of the secondpolymeric solution be incapable of dissolving the first polymericsolution.

The solidification rates of the first and second polymeric solutions arecritical for forming the microtube. For example, for a microtube ofabout 100 μm, the solidification of the first polymer (of the firstpolymeric solution) can be within about 30 milliseconds (ms) while thesolidification of the second polymer (of the second polymeric solution)can be within about 10-20 seconds. The solidification may be a result ofpolymerization rate and/or evaporation rate.

According to some embodiments of the invention; the solvent of the firstpolymeric solution evaporates faster than the solvent of secondpolymeric solution (e.g., the solvent of the first polymeric solutionexhibits a higher vapor pressure than the solvent of the secondpolymeric solution).

According to some embodiments of the invention, the rate of evaporationof the solvent of the first polymeric solution is at least about 10times faster than that of the solvent of the second polymeric solution.The evaporation rate of the solvent of the first polymeric solution canbe at least about 100 times faster or at least about 1000 times fasterthan the evaporation rate of the solvent of second polymeric solution.For example, the evaporation of chloroform is significantly faster thanthe evaporation of an aqueous solution (water) due to the high vaporpressure at room temperature of the chloroform (195 mmHg) vs. that ofthe aqueous solution (23.8 mmHg).

When selecting a solvent of the second polymeric solution which isincapable of dissolving the first polymeric solution (i.e., anon-solvent of the first polymeric solution), the polymer of the firstpolymeric solution can solidify (e.g., through precipitation) and form astrong microtube shell which does not collapse, and is characterized byan even thickness. According to some embodiments of the invention, thefirst polymeric solution (e.g., the solvent of the first polymer) issubstantially immiscible in the solvent of the second polymericsolution.

The solvent of the second polymeric solution may evaporate while thepolymer (of the second polymeric solution) forms a thin layer on theinternal surface of the shell.

According to some embodiments of the invention, the solvent of thesecond polymeric solution is capable of evaporating through the internalsurface of the shell.

The flow rates of the first and second polymeric solutions can determinethe microtube outer and inner diameter and thickness of shell.Non-limiting examples of microtubes generated by electrospinning usingdifferent flow rates are shown in Table 1 hereinbelow.

TABLE 1 Effect of the flow rates of the two polymeric solutions duringelectrospinning on microtube diameter and thickness of shell System:First polymeric R solution/ Outer d Electro- Second Flow Fiber Shell Vstatic System polymeric rates radius thickness Voltage field No.solution (ml/hr) (μm) (μm) (kV) kV/cm M5 First 4 3.0-4.5 0.5 ± 0.1 8.50.43 polymeric solution Second 0.5 polymeric solution M10 First 102.3-4.0 1.0 ± 0.1 8 0.5 polymeric solution Second 0.3 polymeric solutionM11 First 10 3-6 1.0 ± 0.1 9 0.56 polymeric solution Second 2 polymericsolution Table 1: Electrospinning was performed with the followingsolutions: First polymeric solution (for forming the shell) was 10% PCLin CHCl₃/DMF (8:2 weight/weight); Second polymeric solution (for formingthe coat) was 4% PEO in H₂O/EtOH (6:4, weight/weight). PCL used was PCL80 K; PEO used was PEO 600 K. The temperature during electrospinning was22-26° C. The relative humidity during electrospinning was 58%, 52% and53% for systems M5, M10 and M11, respectively. The flow rates weremeasured in milliliter per hour (ml/hr); the outer microtube radius (R)and the shell thickness (d) were measured in microns (μm). The voltagewas measured in kilo volt (kV), and the electrostatic field was measuredin kV per centimeter (cm). The resulting tubes were hollow (good tubesin systems M5 and M11, and mostly good in system M10).

As used herein the phrase “polymeric solution” refers to a solublepolymer, i.e., a liquid medium containing one or more polymers,co-polymers or blends of polymers dissolved in a solvent. The polymerused by the invention can be a natural, synthetic, biocompatible and/orbiodegradable polymer.

The phrase “synthetic polymer” refers to polymers that are not found innature, even if the polymers are made from naturally occurringbiomaterials. Examples include, but are not limited to, aliphaticpolyesters, poly(amino acids), copoly(ether-esters), polyalkylenesoxalates, polyamides, tyrosine derived polycarbonates,poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters,polyoxaesters containing amine groups, poly(anhydrides),polyphosphazenes, and combinations thereof.

Suitable synthetic polymers for use by the invention can also includebiosynthetic polymers based on sequences found in naturally occurringproteins such as those of collagen, elastin, thrombin, fibronectin, orderivatives thereof or, starches, poly(amino acids), poly(propylenefumarate), gelatin, alginate, pectin, fibrin, oxidized cellulose,chitin, chitosan, tropoelastin, hyaluronic acid, polyethylene,polyethylene terephthalate, poly(tetrafluoroethylene), polycarbonate,polypropylene and poly(vinyl alcohol), ribonucleic acids,deoxyribonucleic acids, polypeptides, proteins, polysaccharides,polynucleotides and combinations thereof.

The phrase “natural polymer” refers to polymers that are naturallyoccurring. Non-limiting examples of such polymers include, silk,collagen-based materials, chitosan, hyaluronic acid, albumin,fibrinogen, and alginate.

As used herein, the phrase “co-polymer” refers to a polymer of at leasttwo chemically distinct monomers. Non-limiting examples of co-polymersinclude, polylactic acid (PLA)-polyethyleneglycol (PEG), polyethyleneglycol terephthalate (PEGT)/polybutylene terephthalate (PBT),PLA-polyglycolic acid (PGA), PEG-polycaprolactone (PCL) and PCL-PLA.

As used herein, the phrase “blends of polymers” refers to the result ofmixing two or more polymers together to create a new material withdifferent physical properties.

The phrase “biocompatible polymer” refers to any polymer (synthetic ornatural) which when in contact with cells, tissues or body fluid of anorganism does not induce adverse effects such as immunological reactionsand/or rejections, cellular death and the like. It will be appreciatedthat a biocompatible polymer can also be a biodegradable polymer.

According to some embodiments of the invention, the first and the secondpolymeric solutions are biocompatible.

Non-limiting examples of biocompatible polymers include polyesters (PE),PCL, Calcium sulfate, PLA, PGA, PEG, polyvinyl alcohol, polyvinylpyrrolidone, Polytetrafluoroethylene (PTFE, teflon), polypropylene (PP),polyvinylchloride (PVC), Polymethylmethacrylate (PMMA), polyamides,segmented polyurethane, polycarbonate-urethane and thermoplasticpolyether urethane, silicone-polyether-urethane,silicone-polycarbonate-urethane collagen, PEG-DMA, alginate,hydroxyapatite and chitosan, blends and copolymers thereof.

The phrase “biodegradable polymer” refers to a synthetic or naturalpolymer which can be degraded (i.e., broken down) in the physiologicalenvironment such as by proteases or other enzymes produced by livingorganisms such as bacteria, fungi, plants and animals. Biodegradabilitydepends on the availability of degradation substrates (i.e., biologicalmaterials or portion thereof which are part of the polymer), thepresence of biodegrading materials (e.g., microorganisms, enzymes,proteins) and the availability of oxygen (for aerobic organisms,microorganisms or portions thereof), lack of oxygen (for anaerobicorganisms, microorganisms or portions thereof) and/or other nutrients.Examples of biodegradable polymers/materials include, but are notlimited to, collagen (e.g., Collagen I or IV), fibrin, hyaluronic acid,polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL),polydioxanone (PDO), trimethylene carbonate (TMC), polyethyleneglycol(PEG), collagen, PEG-DMA, alginate, chitosan copolymers or mixturesthereof.

According to some embodiments, the polymeric solution can be made of oneor more polymers, each can be a polymer or a co-polymer such asdescribed hereinabove.

According to some embodiments of the invention, the polymeric solutionof the invention is a mixture of at least one biocompatible polymer anda co-polymer (either biodegradable or non-biodegradable).

According to some embodiments of the invention, the first polymericsolution for forming the shell can be made of a polymer such as poly(e-caprolactone) (PCL), polyamide, poly(siloxane), poly(silicone),poly(ethylene), poly(vinyl pyrrolidone), poly(2-hydroxyethylmethacrylate), poly(N-vinyl pyrrolidone), poly(methylmethacrylate), poly(vinyl alcohol), poly(acrylic acid), poly(vinylacetate), polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethyleneglycol), poly(methacrylic acid), polylactide, polyglycolide,poly(lactide-coglycolide), polyanhydride, polyorthoester,poly(carbonate), poly(acrylo nitrile), poly(ethylene oxide),polyaniline, polyvinyl carbazole, polystyrene, poly(vinyl phenol),polyhydroxy acid, poly(caprolactone), polyanhydride,polyhydroxyalkanoate, polyurethane, collagen, albumin, alginate,chitosan, starch, hyaluronic acid, and blends and copolymers thereof.

According to some embodiments of the invention, the second polymericsolution for forming the coat over the internal surface of the shell canbe made of a polymer such as poly(acrylic acid), poly(vinyl acetate),polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethylene glycol),poly(methacrylic acid), polylactide, polyglycolide,poly(lactide-coglycolide), polyanhydride, polyorthoester,poly(carbonate), poly(ethylene oxide), polyaniline, polyvinyl carbazole,polystyrene, poly(vinyl phenol), polyhydroxy acid, alginate, starch,hyaluronic acid, and blends and copolymers thereof.

During the formation of the microtube shell (e.g., following thesolidification of the first polymeric solution) the second polymericsolution flows within the internal surface of the shell.

According to some embodiments of the invention, the second polymericsolution is selected capable of wetting the internal surface of theshell.

Various polymeric solutions which are known in the art as capable ofwetting other polymeric surfaces (for forming the shell) can be used.Following is a non-limiting list of pairs of polymeric solutions inwhich the second polymeric solution is capable of wetting the internalsurface of the shell formed by the first polymeric solution.

TABLE 2 Pairs of polymeric solutions for producing the microtube of theinvention First polymeric solution forming the Second polymeric solutioncapable of shell wetting the internal surface of the shell 10% poly(e-caprolactone) (PCL); in 4% poly(ethylene oxide) (PEO); in waterchloroform (CHCl₃) and (H₂O) and ethanol (60:40 by weight)dimethylforamide (DMF) (80:20 by weight) Nylon 6,6 in formic acid 7 to12 wt % 4% poly(ethylene oxide) (PEO); in water (H₂O) and ethanol (60:40by weight) Poly(L-lactide-co-glycolide) (PLGA 4% poly(ethylene oxide)(PEO) in water 10:90) in hexafluroisopropanol (HFIP) (H₂O) and ethanol(60:40 by weight) concentrations ranging from 2 to 7 weight % solution.Poly(L-lactide-co-glycolide) (PLGA 4% poly(ethylene oxide) (PEO); inwater 15:85) hexafluroisopropanol (HFIP) (H₂O) and ethanol (60:40 byweight) concentrations ranging from 2 to 7 weight % solution.poly(lactide-co-glycolide) (PLGA; 4% poly(ethylene oxide) (PEO); inwater 1-lactide/glycolide_50/50) (H₂O) and ethanol (60:40 by weight)1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) concentrations ranging from 2to 7 weight % solution. polyglycolide (PGA) in chloroform 3-10 9%poly(vinyl alcohol) (PVA); in water weight % solution. and ethanol(50:50 by weight) poly(L-lactide) (PLA) in chloroform 3-10 9% poly(vinylalcohol) (PVA); in water weight % solution. and ethanol (50:50 byweight) Segmented polyurethane in DMF and 9% poly(vinyl alcohol) (PVA);in water THF (80:20 by weight) and ethanol (50:50 by weight)Polyurethane in DMF and 9% poly(vinyl alcohol) (PVA); in watertetrahydrofuran, THF (80:20 by weight) and ethanol (50:50 by weight)PLGA (poly lactic-co-glycolic acid); in 9% poly(vinyl alcohol) (PVA); inwater chloroform and DMSO (dimethyl and ethanol (50:50 by weight)sulfoxide) in chloroform and DMSO (80:20 by weight). 10% PCL inCHCl₃/DMF (80:20 by 6% PEO in H₂O/EtOH (60:40 by weight) weight) 9% PCLin CHCl₃/DMSO (90:10 by 7% PEO in H₂O weight) 10% PCL in CHCl₃/DMF(80:20 by 9% PVA in ethanol/water (50:50 by weight) weight) 10% PCL 80 KCHCl₃:DMF (90:10 by 4% (w/w) PEO 600 K; in ethanol:H₂O weight) (26:74 byweight) 10% PCL 80 K + 1% PEG 6 K 4% (w/w) PEO 600 K; in ethanol:H₂OCHCl₃:DMF (90:10 by weight) (26:74 by weight) Table 2. The polymersforming the solutions and the solvents are provided by weight ratios,i.e., a weight/weight (w/w) ratio.

According to some embodiments of the invention, the first and the secondpolymeric solutions are selected from the group consisting of: 10% poly(e-caprolactone) (PCL) in chloroform (CHCl₃) and dimethylforamide (DMF)(80:20 by weight) as the first polymeric solution and 4% poly(ethyleneoxide) (PEO) in water (H₂O) and ethanol (60:40 by weight) as the secondpolymeric solution, 10% PCL in CHCl₃ and DMF (80:20 by weight) as thefirst polymeric solution and 6% PEO in water and ethanol (60:40 byweight) as the second polymeric solution, 9% PCL in CHCl₃ and DMF (90:10by weight) as the first polymeric solution and 7% PEO in water as thesecond polymeric solution, 10% PCL in CHCl₃ and DMF (80:20 by weight) asthe first polymeric solution and 9% poly(vinyl alcohol) (PVA) in waterand ethanol (50:50 by weight) as the second polymeric solution and 10%PCL in CHCl₃ and DMF (90:10 by weight) as the first polymeric solutionand 4% (w/w) PEO in ethanol:H₂O (26:74 by weight) as a second polymericsolution.

To enable a flow of a liquid-of-interest within the microtube, i.e.,along the coat polymer covering the internal surface of the shell (whichoriginates from the second polymer solution), the surface (thin film)formed by the coat polymer should be designed such that it can be wettedby the liquid-of-interest. The ability to wet (wettability) polymerfilms by liquids is known in the art. For example, silicone oil or watercan wet a surface made of a PEO polymer. It will be appreciated that thewettability of the coat polymer covering the internal surface of theshell can be controlled (e.g., improved) for example by attachingfunctional groups such as hydroxyl groups (OH) which increase thehydrophilicity of the coat by a plasma treatment [see Thurston R M, ClayJ D, Schulte M D, Effect of atmospheric plasma treatment on polymersurface energy and adhesion, Journal of Plastic Film & Sheeting 23 (1):63-78 January 2007; which is incorporated within by reference].

As is further discussed hereinabove and in the Examples section whichfollows, for certain applications the microtube shell may comprisepores, thus creating a “breathing” tube. Methods of forming “breathing”microtube microtubes with pores in the shell thereof) are described inPCT/IB2007/054001 to the present inventors, which is fully incorporatedherein by reference. Briefly, “breathing” tubes can be formed by theinclusion of a high percent (e.g., at least 80%) of a volatile componentsuch as tetrahydrofuran (THF), chloroform, acetone, or trifluoroethanol(TFE) in the first polymeric solution forming the shell, and/or by theinclusion of a water-soluble polymer such as polyethylene glycol (PEG)in the first polymeric solution forming the shell so that the firstpolymeric solution comprises a blend of polymers in which one iswater-soluble and the other is water-insoluble (e.g., a blend of PEG andPCL). Alternatively, “breathing” microtubes can be formed by inducingpores in the shell after the completion of the electrospinning process,essentially as described in PCT WO 2006/106506 to the present inventors,which is fully incorporated herein by reference, such as by passing anelectrical spark or a heated puncturing element through the electrospunshell, or by using a pulsed or continuous laser beam through theelectrospun shell.

According to some embodiments of the invention, the first polymericsolution comprises PEG for inducing pores in the shell. For example, togenerate pores greater (>) than 150 nm in diameter, the first polymericsolution may include about 4% PEG MW 35 kDa. Similarly, to generatepores smaller (<) than 150 nm in diameter, the first polymeric solutionmay include about 2% PEG MW 6 kDa.

The microtube shell of the invention can be designed such that itenables the passage of certain molecules (e.g., a substrate of anenzyme) while preventing the passage of other molecules (e.g., a certainenzyme), depending on the geometry (pore size) and/or the electricalcharge of the molecules with respect to the geometry (length andradius), surface energy, electrical charge of the nanopore(s) of theshell, and the viscosity and surface tension of the liquid containingthe molecules (e.g., the substrate of the enzyme). In addition, theporosity and pore size of the shell can control the release of themolecule-of-interest which is attached to the microtube. For example, ahigher porosity and/or pore size can result in increased rate of releaseof the molecule-of-interest.

Alternatively, the microtube shell can be made such that it preventsdiffusion or any passage of the molecule-of-interest therethrough (i.e.,substantially devoid of pores, or with pores smaller than themolecule-of-interest).

As mentioned, the second polymeric solution comprises themolecule-of-interest. Such a molecule (or molecules) can be anynaturally occurring or synthetic molecule such as a polypeptide, apolynucleotide, a carbohydrate or a polysaccharide, a lipid, a drugmolecule, a small molecule (e.g., a nucleotide base, an amino acid, anucleotide, an antibiotic, a vitamin or a molecule which is smaller than0.15 kDa), or any combination thereof. The molecule-of-interest can beproduced by recombinant DNA technology or by known synthesis methodssuch as solid phase.

According to some embodiments of the invention, the molecule-of-interestcomprises a polypeptide such as an enzyme. Such polypeptides (e.g.,enzymes) can be naturally occurring (e.g., mammals such as primates,rodents and Homo sapiens, plants, fungi, protozoa, bacteria and viruses)or synthetic (e.g., derived from in vitro evolution) and can be selectedaccording to the desired application.

The following non-limiting list of enzymes can be attached to themicrotube of the invention: DNA polymerase (EC 2.7.7.7), DNase (EC3.1.11.4), RNA polymerase (EC 2.7.7.6), DNA ligase (EC 6.5.1.1), RNAligase (EC 6.5.1.3), alcohol dehydrogenase (EC 1.1.1.1), homoserinedehydrogenase (EC 1.1.1.3), acetoin dehydrogenase (EC 1.1.1.5), glyceroldehydrogenase (EC 1.1.1.6), L-xylulose reductase (EC 1.1.1.10),L-arabinitol 2-dehydrogenase (EC 1.1.1.13), L-iditol 2-dehydrogenase (EC1.1.1.14), mannitol-1-phosphate 5-dehydrogenase (EC 1.1.1.17), mannitol2-dehydrogenase (EC 1.1.1.138), glucose oxidase (EC 1.1.3.4), L-sorboseoxidase (EC 1.1.3.11), lactate-malate transhydrogenase (EC 1.1.99.7),formaldehyde dehydrogenase (EC 1.2.1.1), aryl-aldehyde dehydrogenase (EC1.2.1.29), aldehyde oxidase (EC 1.2.3.1), pyruvate synthase (EC1.2.7.1), cortisone α-reductase (EC 1.3.1.4), lathosterol oxidase (EC1.33.2), D-proline reductase (EC 1.4.4.1), dihydrofolate reductase (EC1.5.1.3), methylenetetrahydrofolate reductase (NADPH) (EC 1.5.1.20),cystine reductase (NADH) (EC 1.6.1.4), cob(II)alamin reductase (EC1.6.99.9), sulfite reductase (EC 1.8.1.2), cytochrome-c oxidase (EC1.9.3.1), NADH peroxidase (EC 1.11.1.1), homogentistate 1,2-dioxygenase(EC 1.13.11.5), Photinus-luciferin 4-monooxygenase (1.13.12.7),anthranilate 3-monooxygenase (EC 1.14.13.35), steroid 9α-monooxygenase(EC 1.14.99.25), mercury(II) reductase (EC 1.16.1.1), nicotinamideN-methyltransferase (EC 2.1.1.1), thymidylate synthase (EC 2.1.1.45),site-specific DNA-methyltransferase (adenine-specific) (EC 2.1.1.72),tryptophan 2-C-methyltransferase (EC 2.1.1.106), glycineformininotransferase (EC 2.1.2.4), aspartate carbamoyltransferase (EC2.1.3.2), transaldolase (EC 2.2.1.2), arylamine N-acetyltransferase (EC2.3.1.5), arginine N-succinyltransferase (EC 2.3.1.109), phosphorylase(EC 2.4.1.1), glycosaminoglycan galactosyltransferase (EC 2.4.1.74),thymidine phosphorylase (EC 2.4.2.4), β-galactosideα-2,6-sialyltransferase (EC 2.4.99.1), galactose-6-sulfurylase (EC2.5.1.5), aspartate transaminase (2.6.1.1), hexokinase (EC 2.7.1.1),choline kinase (EC 2.7.1.32), acetate kinase (EC 2.7.2.1), creatinekinase (EC 2.7.3.2), adenylate kinase (EC 2.7.4.3), nucleotidepyrophosphokinase (EC 2.7.6.4), sulfate adenylyltransferase (ADP) (EC2.7.7.5), aryl sulfotransferase (EC 2.8.2.1), carboxylesterase(3.1.1.1), acetyl-CoA hydrolase (EC 3.1.2.1), alkaline phosphatase(3.1.3.1), phosphodiesterase I (EC 3.1.4.1), dGTPase (EC 3.1.5.1),steryl-sulfatase (EC 3.1.6.2), exodeoxyribonuclease I (EC 3.1.11.1),ribonuclease T1 (EC 3.1.27.3), α-amylase (EC 3.2.1.1), purinenucleosidase (EC 3.2.2.1), epoxide hydrolase (EC 3.3.2.3), lysylaminopeptidase (EC 3.4.11.15), carboxypeptidase A2 (EC 3.4.17.15),trypsin (EC 3.4.21.4), glutaminase (EC 3.5.1.2), barbiturase (EC3.5.2.1), ATP deaminase (EC 3.5.4.18), inorganic pyrophosphatase (EC3.6.1.1), oxaloacetase (EC 3.7.1.1), oxalate decarboxylase (EC 4.1.1.2),mandelonitrile lyase (EC 4.1.2.10), isocitrate lyase (4.1.3.1), fumaratehydratase (EC 4.2.1.2), pectate lyase (EC 4.2.2.2), histidineammonia-lyase (EC 4.3.1.3), cyanate lyase (4.3.99.1), cysteine lyase (EC4.4.1.10), DDT-dehydrochlorinase (EC 4.5.1.1), adenylate cyclase (EC4.6.1.1), alanine racemase (5.1.1.1). tartrate epimerase (EC 5.1.2.5),retinal isomerase (EC 5.2.1.3), L-rhamnose isomerase (EC 5.3.1.14),prostaglandin-D synthase (EC 5.3.99.2), phosphoglucomutase (EC 5.4.2.2),lanosterol synthase (EC 5.4.99.7), DNA topoisomerase (EC 5.99.1.2),tyrosine-tRNA ligase (EC 6.1.1.1), acetate-CoA ligase (EC 6.2.1.1),acetylcholinesterase (EC 3.1.1.7), butyrylcholinesterase (EC 3.1.1.8)and glutathione synthase (EC 6.3.2.3).

According to an embodiment of the invention, the enzyme which isattached to the microtube is alkaline phosphatase (e.g., SEQ ID NO:1 or8; EC 3.1.3.1) or beta-galactosidase (e.g., SEQ ID NO:2 or 9; EC3.2.1.23).

The term “polynucleotide” as used herein refers to a single stranded ordouble stranded oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimetics thereof. This term includespolynucleotides composed of naturally-occurring bases, sugars andcovalent internucleoside linkages (e.g., backbone) as well aspolynucleotides having non-naturally-occurring portions which functionsimilarly to respective naturally-occurring portions.

The polynucleotide which is attached to the microtube of the inventioncan be generated according to any oligonucleotide synthesis method knownin the art such as enzymatic synthesis, liquid phase or solid phasesynthesis (using a commercially available equipment from, for example,Applied Biosystems). Equipment and reagents for executing solid-phasesynthesis are commercially available from, for example, AppliedBiosystems. Any other means for such synthesis may also be employed; theactual synthesis of the oligonucleotides is well within the capabilitiesof one skilled in the art and can be accomplished via establishedmethodologies as detailed in, for example, “Molecular Cloning: Alaboratory Manual” Sambrook et al., (1989); “Current Protocols inMolecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel etal., “Current Protocols in Molecular Biology”, John Wiley and Sons,Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”,John Wiley & Sons, New York (1988) and “Oligonucleotide Synthesis” Gait,M. J., ed. (1984) utilizing solid phase chemistry, e.g. cyanoethylphosphoramidite followed by deprotection, desalting and purification byfor example, an automated trityl-on method or HPLC. Liquid phasesynthesis of oligonucleotides can be performed using methods known inthe art (see for example, Bonora G M, et al., 1998, Biol. Proced.Online. 1: 59-69; Padiya K J and Salunkhe M M., 2000, Bioorg. Med. Chem.8: 337-42). It will be appreciated that for the preparation of multiplelabeled polynucleotides, a large scale oligonucleotide synthesis can beutilized essentially as described elsewhere (Anderson N G. et al., ApplBiochem Biotechnol. 1995 July-September; 54(1-3):19-42; Rahmann S., ProcIEEE Comput Soc Bioinform Conf. 2002; 1:54-63).

Additionally or alternatively, the polynucleotide which is attached tothe microtube of the invention can be generated by recombinant DNAtechniques using any known DNA replication or transcription system(e.g., using bacterial cells, eukaryotic cells).

As mentioned, the molecule-of-interest can be a drug molecule. Such adrug can be any synthetic, chemical or biological molecule.

Non-limiting examples of biological drug molecules include antisenseoligonucleotides, Ribozymes, DNAzymes, siRNA, receptor agonists,antagonists, hormones, growth factors and antibodies. Non-limitingexamples of which chemical drug molecules include chemotherapy agents,Paclitaxel (Taxol®), radiation seed particles (e.g., see HypertextTransfer Protocol://World Wide Web (dot) oncura (dot) corn), as well asnatural or synthetic vitamins.

The molecule-of-interest which is attached to the microtube of theinvention can be labeled. Such a label can be an intrinsic property ofthe molecule-of-interest (e.g., as in the case of green fluorescentprotein) or can be a label which is attached to the molecule-of-interestusing known methods. For example, the label can be a fluorescentlabeling in which a fluorophore (i.e., an entity which can be excited bylight to emit fluorescence) or a radio-isotope is conjugated via alinker or a chemical bond to the molecule-of-interest. Alternatively,the molecule-of-interest can be indirectly labeled via a covalentlyconjugated enzyme (e.g., horse radish peroxidase) and a covalentlyconjugated substrate (e.g., o-phenylenediamine) which upon interactiontherebetween yield a colorimetric or fluorescent color.

The molecule-of-interest can also comprise a member of an affinity pair,which is capable of reversibly or non-reversibly binding with highaffinity (e.g., less than 10⁻⁷ M, e.g., less than 10⁻⁸ M, less than10⁻⁹, less than 10⁻¹⁰ M) to a specific molecule. For example, theaffinity pair can be an enzyme-substrate pair, a polypeptide-polypeptidepair (e.g., a hormone and receptor, a ligand and receptor, an antibodyand an antigen, two chains of a multimeric protein), a polypeptide-smallmolecule pair (e.g., avidin or streptavidin with biotin,enzyme-substrate), a polynucleotide and its cognate polynucleotide suchas two polynucleotides forming a double strand (e.g., DNA-DNA, DNA-RNA,RNA-DNA), a polypeptide-polynucleotide pair (e.g., a complex formed of apolypeptide and a DNA or RNA e.g., aptamer), a polypeptide-metal pair(e.g., a protein chelator and a metal ion), a polypeptide and acarbohydrate (leptin-carbohydrate), and the like.

The molecule-of-interest, which is comprised within the second polymericsolution, can be attached to the coat over the internal surface of theshell. For example, as shown in FIG. 5 and described in Example 3 of theExamples section which follows, most of the n-GAL enzymatic activity wasdetected inside the microtube, demonstrating the attachment of theenzyme to the coat over the internal surface of the shell.

During the electrospinning process some molecules-of-interest which arecomprised within the second polymeric solution may migrate to the outersurface of the shell (i.e., mixed with the first polymeric solution)depending on their charge state, size and geometry. For example, asshown in FIG. 3 and described in Example 2 of the Examples section whichfollows, some of the alkaline phosphatase activity was detected in therinsing buffer of the microtube.

According to some embodiments of the invention attachment of themolecule-of-interest is performed following microtube formation. Forexample, the microtube can be soaked with a solution containing themolecule-of-interest. The molecule-of-interest can diffuse through theshell pores and enter the inner lumen of the microtube. In addition, themicrotube can be covalently attached to the molecule-of-interest (e.g.,via SH groups).

Regardless of the method of production, the present invention provides amicrotube which comprises an electrospun shell, an electrospun coat overan internal surface of the shell and a molecule-of-interest attached tothe microtube.

As used herein, the phrase “electrospun shell” refers to a hollowelement of a tubular shape, made of one or more polymers, produced bythe process of electrospinning as detailed above.

As used herein the phrase “electrospun coat” refers to a thin layercovering the internal surface of the shell of the microtube of theinvention which is made of one or more polymers by the process ofelectrospinning as detailed above.

One of ordinary skill in the art will know how to distinguish anelectrospun object from objects made by means which do not compriseelectrospinning by the high orientation of the macromolecules, the skin(e.g., shell) morphology, and the typical dimensions of the microtubewhich are unique to electrospinning.

The microtube of the invention can be an individual (e.g., single orseparated) microtube or can form part of a plurality (e.g., an alignedarray) of microtubes which can be either connected to each other orseparated (as single, not-connected microtubes).

For the production of a single microtube a fork like clip is attached tothe edge of the rotating disk. The disk is rotated for 1-2 seconds andindividual microtubes are formed between the sides of the clip. In asimilar way individual electrospun fibers were collected (see E.Zussman, M. Burman, A. L. Yarin, R. Khalfin, Y. Cohen, “TensileDeformation of Electrospun Nylon 6,6 Nanofibers,” Journal of PolymerScience Part B: Polymer Physics, 44, 1482-1489, 2006, hereinincorporated by reference in its entirety).

Alternatively, when using a rotating collector, a plurality ofmicrotubes can be formed and collected on the edge of the collector asdescribed elsewhere for electrospun fibers (A. Theron, E. Zussman, A. L.Yarin, “Electrostatic field-assisted alignment of electrospunnanofibers”, Nanotechnology J., 12, 3: 384-390, 2001; hereinincorporated by reference in its entirety).

The plurality of microtubes can be arranged on a single layer, oralternatively, the plurality of microtubes define a plurality of layershence form a three dimensional structure. The microtubes can have ageneral random orientation, or a preferred orientation, as desired. Forexample, when the fibers are collected on a cylindrical collector suchas a drum, the microtubes can be aligned predominantly axially orpredominantly circumferentially. Different layers of the electrospunmicrotubes can have different orientation characteristics. For example,without limiting the scope of the present invention to any specificordering or number of layers, the microtubes of a first layer can have afirst predominant orientation, the microtubes of a second layer can havea second predominant orientation, and the microtubes of third layer canhave general random orientation.

The microtube of the invention can be available as a dry fibrous mat(s)(e.g., as spun dry microtubes) or as a wetted mat(s) (e.g., followingimmersing or filling the microtube with a liquid).

The microtube of the invention which is attached to themolecule-of-interest may be configured as or in a microfluidics device.“Lab-on-a-chip” is described in a series of review articles [see forexample, Craighead, H. Future lab-on-a-chip technologies forinterrogating individual molecules. Nature 442, 387-393 (2006); deMello,A. J. Control and detection of chemical reactions in microfluidicsystems. Nature 442, 394-402 (2006); El-Ali, J., Sorger, P. K. & Jensen,K. F. Cells on chips. Nature 442, 403-411 (2006); Janasek, D., Franzke,J. & Manz, A. Scaling and the design of miniaturized chemical-analysissystems. Nature 442, 374-380 (2006); Psaltis, D., Quake, S. R. & Yang,C. H. Developing optofluidic technology through the fusion ofmicrofluidics and optics. Nature 442, 381-386 (2006); Whitesides, G. M.The origins and the future of microfluidics. Nature 442, 368-373 (2006);Yager, P. et al. Microfluidic diagnostic technologies for global publichealth. Nature 442, 412-418 (2006)] each of which is fully incorporatedherein by reference].

According to some embodiments of the invention, the liquid which fillsin, flows in or surrounds the microtube enables the desorption(detachment) of the molecule-of-interest from the microtube (e.g., fromthe polymer included in the coat over the internal surface of theshell). According to of some embodiments of the invention the desorptionprocess facilitates the interaction between the molecule-of-interest anda substrate. According to some embodiments of the invention thedesorption process enables the flow and/or the release of themolecule-of-interest within and/or from the microtube.

According to some embodiments of the invention, the molecule-of-interestwhich is attached to the microtube of the invention remains active,maintains the activity, or at least a portion thereof, which itpossessed prior to the attachment (e.g., of the samemolecule-of-interest before electrospinning, or when not-attached to themicrotube). The term “activity” as used herein refers to any of acatalytic activity, kinetics, and/or affinity to a substrate, a ligandor an affinity member of the molecule. Such an activity can be anybiological activity such as catalysis, binding (with a specificaffinity), hybridization, chelation, degradation, synthesis, catabolism,hydrolysis, polymerization, transcription, and the like.

As used herein the phrase “at least a portion of the activity” refers toat least about 10%, at least about 20-50%, e.g., more than about 50%,e.g., more than about 60%, e.g., more than about 70%, e.g., more thanabout 75%, e.g., more than about 80%, e.g., more than about 90%, e.g.,more than about 95% of the activity which the molecule-of-interestpossessed prior to the attachment to the microtube.

For example, as mentioned before and described in the Examples sectionwhich follows, the enzymes contained within the microtubes preserved thespecific activity to their substrates at a kinetic which is comparable(i.e., within the same order of magnitude) to that of the enzyme in thepolymeric solution prior to electrospinning.

The microtube of the invention which is attached to an activemolecule-of-interest can be used in various applications which requirethe attachment of active molecules (e.g., enzymes, DNA, RNA) to asupport and optionally also the controlled release therefrom.

According to some embodiments of the invention, the microtube of theinvention is attached to more than one type of molecule-of-interest. Thecombination of molecules can be selected according to the intended use.For example, several molecules (e.g., enzymes) which are involved incomplex reactions (e.g., processing of a substrate or a mixture ofsubstrates) can be used.

Thus, according to an aspect of the invention, there is provided amethod of processing a substrate-of-interest. The method is effected bycontacting the substrate-of-interest with the microtube of theinvention, wherein the molecule-of-interest is capable of processing thesubstrate, thereby processing the substrate-of-interest.

As used herein the term “processing” refers to a catalytic activityperformed by the molecule-of-interest which is attached to the microtubeon its cognate substrate. According to some embodiments of theinvention, such a process can concomitantly incorporate of thesubstrate-of-interest in a synthesis reaction catalyzed by themolecule-of-interest.

For example, the microtube of some embodiments the invention can be usedas a micro-reactor (e.g., bioreactor) for chemical transition reactionsrequiring high concentrations of several enzymes. As described inExample 4 of the Examples section which follows and schematicallyillustrated in FIG. 9, the microtube of the invention can be attached tocertain molecules (enzymes in this case), which together catalyze amulti-step synthesis reaction (e.g., cascade) which converts an initialsubstrate (e.g., compound A) to an end-product (e.g., compound E). Asmentioned, the selective shell of the microtube can be designed suchthat it prevents the leakage (escape by diffusion) of the intermediatecompounds (e.g., compounds B, C and D) therethrough and thus enablessufficiently high concentrations of such compounds as needed for thesynthesis of the end product. The local concentration of theintermediate molecules formed from the initial substrate (entrapped atthe time of spinning or externally added after electro spinning to theformed microtube) are about 2-10 orders of magnitude greater than theconcentrations formed in an open system. Thus, the microtube of someembodiments of the invention exhibits a great kinetic advantage inmulti-step reactions as compared to an open system. Microtubes are inthis way similar to living cells which function on the same principle.

For example, to synthesize an indole-glycerol phosphate, an intermediatecompound in tryptophan synthesis within cells, a microtube of someembodiments of the invention can be attached to the enzymesanthranilate-phosphoribosyl transferase (EC 2.4.2.18),phosphoribosylanthranilate isomerase (EC 5.3.1.24) andindole-3-glycerol-phosphate synthase (EC 4.1.1.18), and the reactioncommences when anthranilate and phosphoribosyl-pyrophosphate interactwith the attached enzymes. The substrates (anthranilate andphosphoribosyl-pyrophosphate) can be either added externally to thereaction medium (within which the microtube is placed) or can beattached to the microtube by mixing them within the second polymericsolution. When the substrates are supplied externally, pores of about2-20 nm in diameter should exist in the shell to allow diffusion ofanthranilate and phosphoribosyl-pyrophosphate therethrough.

According to some embodiments of the invention, such a process can bethe incorporation of the substrate-of-interest in a catabolism reactioncatalyzed by the molecule-of-interest.

A catabolism reaction can be the degradation (e.g., by hydrolysis) of atoxic molecule for the purpose of detoxification (e.g., detoxifyingwater) or decomposition of an unwanted molecule. Examples include, butare not limited to, the removal of the chlorine entity from atrazine(see FIG. 10) and the degradation of cyanide resulting from silvermining.

According to an aspect of the invention, there is provided a method ofdepleting a molecule from a solution. The method is effected bycontacting the solution with the microtube of the invention, wherein themember of the affinity pair (which is attached to the microtube) isselected capable of binding the molecule (which is to be removed),thereby depleting the molecule from the solution.

According to an embodiment of the invention, the method furthercomprising collecting the solution following the contacting.

As used herein the phrase “depleting” refers to removing an amount e.g.,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, e.g., 99%, e.g., 100%of the molecule from the solution.

According to some embodiments of the invention, the depletion (removal)of the molecule from the solution is effected within a short timeperiod, such as within minutes (e.g., 1-30 minutes), hours (e.g., 1-10hours) or several days (e.g., 1-5 days).

As used herein the phrase “contacting” refers to enabling theinteraction between the molecule and the member of the affinity pair,which is attached to the microtube, for a time period which issufficient for depleting the molecule from the solution. Such a contactcan take place while the solution is passing through (e.g., viacapillary forces) the end(s) of the hollow structure of the microtubeand/or through the shell pores. Additionally or alternatively, such acontact between the molecule and the member of the affinity pair cantake place by incubating the microtube in the solution (e.g., by placingthe microtube in a container including the solution).

The solution can be any water-based solution which includes inorganic ororganic molecules, such as a biological sample or a sample from anon-living source such as stream or ocean waters. As used herein thephrase “biological sample” refers to any sample derived from a livingorganism such as plant, bacteria or mammals, and can include cells oralternatively be cell-free (i.e., include only a biological fluid). Forexample, a biological sample of an individual can include body fluidssuch as blood or components thereof (e.g., white blood cells, red bloodcells, coagulation factors, leukocytes, neutrophils, serum, plasma),cerebrospinal fluid, urine, lymph fluids, and various externalsecretions of the respiratory, intestinal and genitourinary tracts,tears, saliva, milk, amniotic fluid and chorionic villi, a tissuebiopsy, a tissue section, a malignant tissue, and the like. The samplecan be derived from the individual and be further tested in vitro or exvivo, or alternatively, can be not physically removed from the subject(e.g., for in situ detection and/or diagnosis).

According to some embodiments of the invention, the solution is anaqueous solution such as a drinking water, a groundwater and/or anindustrial waste water. According to some embodiments of the invention,the microtube of the invention forms part of an aqueous system designedfor treatment of the aqueous solution (e.g., for depleting, eliminatingor removing toxic moieties therefrom).

For example, to remove a certain metal ion (e.g., copper, gold, nickel,zinc, lead, mercury, cadmium, silver, iron, manganese, palladium, andplatinum) from water, the microtube of the invention can be attached toa water soluble ethylene dichloride ammonia polymer, which containsdithiocarbamate salt groups and is capable of chelating the metal ion(U.S. Pat. No. 5,346,627). Thus, by contacting the water with themicrotube the ethylene dichloride ammonia polymer binds to the metal ionand removes it from the water. Water collected after being in contactwith the microtube is substantially devoid of the metal ion.Alternatively, these metal ions may be removed by attaching a proteinchelator of such metal ions to the microtube.

Alternatively, to remove a ligand (e.g., a hormone, a substrate, aco-factor or a vitamin such as biotin) from a solution containing abiological sample, the microtube can be attached to a polypeptide whichis member of an affinity pair such as an enzyme, a hormone orstreptavidin, and following contacting the solution with the microtube,the ligand remains attached to the microtube while the solution issubstantially devoid of the ligand (e.g., includes less than 0.5%, e.g.,less than 0.1%, e.g., less than 0.01% of the ligand).

According to some embodiments of the invention, the molecule which is tobe removed from the solution comprises an antigen and the member of theaffinity pair comprises the antibody capable of specifically binding theantigen.

For example, the microtube of the invention can be used to remove virusparticles from a blood sample. Briefly, an anti-virus antibody (e.g.,anti-HIV antibodies such as those described in Tullis, R H., et al.,Therapeutic Apheresis and Dialysis, 6: 213-220) can be attached to themicrotube and a blood sample containing the virus particles (e.g., HIVparticles) can be in contact with the microtube such that the virusparticles bind to their respective antibodies and the collected bloodsample (after being in contact with the microtube) is substantiallydevoid of the viral particles.

The member of the affinity pair which is attached to the microtube ofthe invention can be also used to isolate a molecule from a solution.

According to an aspect of the invention, there is provided a method ofisolating a molecule from a solution. The method is effected by: (a)contacting the solution with the microtube of the invention underconditions which allow binding of the molecule to the microtube via themember of the affinity pair which is selected capable of binding themolecule, and (b) eluting the molecule from the microtube, therebyisolating the molecule from the solution.

As used herein the term “isolating” refers to physically separating themolecule from the solution or its other components by binding themolecule to the member of the affinity pair that is attached to themicrotube and eluting the bound molecule therefrom. As used herein theterm “eluting” refers to dissociating the bound molecule from themicrotube. Those of skills in the art are capable of adjusting theconditions required for eluting (e.g., releasing) the molecule from themicrotube and/or separating the molecule from the other member of theaffinity pair.

As is further described in Example 5 of the Examples section whichfollows, the present inventors have envisaged the use of the microtubeof the invention, which is attached to a member of an affinity pair, asa biosensor, for the detection of molecules in a sample. Such abiosensor can be advantageous over known open field biosensors (e.g.,sensors in which the member of the affinity pair is conjugated to asolid support not having a tubular structure, such as a flat support)due to the increased ratio between the size of the microtube surface(which attaches the member of the affinity pair) and the volume of thesample being in contact therewith.

According to an aspect of the invention, there is provided a method ofdetecting a presence of a molecule in a sample. The method is effectedby (a) contacting the sample with the microtube of the invention,wherein the member of the affinity pair is selected capable of bindingthe molecule, and; (b) detecting binding of the molecule by the memberof the affinity pair, thereby detecting the presence of a molecule inthe sample.

As used herein the phrase “detecting binding” refers to identifying achange in the concentration, conformation, spectrum or electrical chargeof the molecule in the sample and/or the member of the affinity pairthat is attached to the microtube following the binding therebetween.Identification of such binding can be performed using methods known inthe art such as following the fluorescence or the color of the sample,radioactivity in the sample, the electrical conductivity of the sampleand the like.

As mentioned hereinabove and described in Example 2 of the Examplessection which follows, the microtube of the invention can release themolecule-of-interest attached thereto (a releasing apparatus).

The microtube of some embodiments the invention (e.g., a microtube madeof biocompatible polymers) can be implanted in a subject in needthereof.

As used herein the phrase a “subject in need thereof” refers to anyanimal subject e.g., a mammal, e.g., a human being which suffers from apathology (disease, disorder or condition) which can be treated by themolecule that is attached to or flows through the microtube of theinvention.

The term “treating” as used herein refers to inhibiting, preventing orarresting the development of a pathology and/or causing the reduction,remission, or regression of a pathology. Those of skill in the art willunderstand that various methodologies and assays can be used to assessthe development of a pathology, and similarly, various methodologies andassays may be used to assess the reduction, remission or regression of apathology.

Methods of implanting grafts such as the microtube of the invention intoa subject are known in the art. For example, the microtube can beimplanted subcutaneously, intradermally, or into any body cavity (e.g.,abdomen), as well as into the vascular system (using e.g., a hollowcatheter delivery system). Alternatively, the microtube of the inventioncan be connected to a body conduit (e.g., a blood vessel such as a veinor an artery) such that it enables the flow of a fluid therethrough.

For example, the microtube of the invention which is capable ofdepleting a molecule from a solution as described above, can beconnected to a blood vessel of the subject. For example, the proximalend of the microtube (or of a plurality of microtubes) can be connectedto a feeding blood vessel and the distal end of the microtube(s) can beconnected to a receiving blood vessel. Such a configuration can be used,for example, for hemodialysis and depletion of a specific molecule(e.g., a virus particle such as HIV, hepatitis virus such as HCV) fromthe blood stream of the subject.

In addition, a microtube which is attached to a drug molecule can beimplanted in a subject in need thereof to thereby release atherapeutically effective amount of the drug to cells of the subject.

As used herein the phrase “therapeutically effective amount” means anamount of the molecule-of-interest (e.g., the drug, the active molecule)effective to prevent, alleviate or ameliorate symptoms of a pathology orprolong the survival of the subject being treated. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art.

For example, in case the molecule-of-interest comprises apolynucleotide, such a polynucleotide can be used in gene therapyapplications to either increase an expression level or activity of adesired polypeptide needed for treating the pathology, or to decrease orinhibit the expression level of a polynucleotide causing the pathology(e.g., antisense technology). Alternatively, the polynucleotide can beused to immunize the subject by inducing an immune responsethereagainst.

Alternatively, in case the molecule-of-interest is a polypeptide, such apolypeptide can be used in a subject in need of polypeptide therapy,such as a subject having a decreased or no activity of the polypeptide(e.g., due to a genetic disease, auto-antibodies, pathogen infection,degeneration or a decrease in tissue functioning), as well as for othertherapeutic applications such as immunization with the polypeptide.Non-limiting examples of pathologies which require polypeptide therapyand can be treated using the microtube of the invention include,metabolic disorders such as phenylketonuria (PKU), Gaucher disease,muscular dystrophy [Duchenne (DMD) and Becker (BMD) MuscularDystrophies], Aceruloplasminemia (an iron metabolic disorder), endocrinediseases such as diabetes, autoimmune diseases such as multiplesclerosis (MS), rheumatoid arthritis (RA), and psoriasis, and variouscancers (e.g., lymphoma).

Following is a non-limiting list of polypeptides which can be attachedto the microtube of the invention in order to treat pathologiesrequiring polypeptide therapy. Phenylalanine hydroxylase [(PAH); GenBankAccession Nos. NM_000277.1 (nucleic acid sequence) and NP_000268.1 (SEQID NO:3; amino acid sequence)] for treating phenylketonuria (PKU),dystrophin [(DMD); GenBank Accession Nos. NM_000109.2 (nucleic acidsequence) and NP_000100.2 (SEQ ID NO:4; amino acid sequence)] fortreating Duchenne (DMD) and Becker (BMD) Muscular Dystrophies,beta-glucosidase [(GBA); GenBank Accession Nos. NM_001005741.1 (nucleicacid sequence) and NP_001005741.1 (amino acid sequence; SEQ ID NO:5)]for treating Gaucher disease, insulin [GenBank Accession Nos.NM_000207.1 (nucleic acid sequence) and NP 000198.1 (amino acidsequence; SEQ ID NO:6)] for treating diabetes, and ceruloplasminferroxidase [(CP); GenBank Accession Nos. NM_000096.1 (nucleic acidsequence) and NP_000087.1 (SEQ ID NO:7; amino acid sequence)] fortreating aceruloplasminemia, CD20 monoclonal antibodies for treatingnon-Hodgkin's lymphoma and autoimmune disease (Yazawa N, et al., 2005,Proc Natl Acad Sci USA. 102:15178-83) and T-cell receptor peptides fortreating of multiple sclerosis (MS), rheumatoid arthritis (RA), andpsoriasis (Vandenbark A A, et al., 2001, Neurochem Res. 26:713-30).

Targeted delivery of a drug molecule to a tissue-of-interest is desiredin various pathologies, especially in cases where the effect of the drugis deleterious to non-diseased tissues or when high concentrations ofdrug molecules are required to achieve a therapeutic effect on thediseased tissue (the tissue-of-interest). Thus, for example it is highlydesired to have a targeted delivery of a chemotherapy agent or aradiation seed particle to the liver in case of hepatic cancer, or anangiogenic factor to coronary blood vessels, heart or carotid bloodvessels in case of ischemia.

According to an embodiment of the invention, for targeted delivery of adrug molecule to a tissue-of-interest via the opening of the microtube(at the targeted tissue), the microtube of the invention is designedsuch that the electrospun shell is semi-permeable (i.e., preventspassage of the drug molecule but enables the penetration of water or aphysiological solution therethrough) and the coat over the internalsurface of the shell is attached to the drug molecule.

Such a microtube can be implanted in a subject such that the distal endof the microtube is implanted in or in close proximity to thetissue-of-interest. As used herein the term “proximity” refers to beingin a cavity defined by the tissue, for example, if the tissue in whichthe drug is released is a blood vessel (artery or vein) the cavity is alumen of such a blood vessel, or if the tissue in which the medicationis released is a heart chamber, then the cavity is an atrium or aventricle. It will be appreciated that the other end of the microtubecan be also implanted in proximity to the tissue-of-interest.Alternatively, the proximal end of the microtube can be either sealedusing e.g., a laser beam to prevent delivery of the drug to undesiredcells/tissues of the subject, or if needed, could be placed outside thebody, or subcutaneously such that the microtube can be replenished withadditional drug molecules using extra thin needles (e.g., which canpenetrate a 5 μm lumen of the microtube).

Once the microtube of some embodiments of the invention (e.g., amicrotube with a semi permeable shell and a drug molecule attached tothe coat over the internal surface of the shell) is implanted in thesubject it can be filled with a physiological fluid (e.g., of thesubject) which is capable of dissolving the water-soluble polymer of thecoat over the internal surface of the shell to thereby release the drugmolecule therefrom. The released drug molecule flows by capillary forceswithin the microtube until reaching the end of the open lumen, which isin proximity of the tissue-of-interest.

If needed, the microtube according to this embodiment of the invention,can be also replenished with additional drug molecules or othermolecules which can increase the effect of the drug molecule released bythe microtube. For example, if the drug molecule attached to themicrotube is an angiogenic factor, a solution saturated with gasses(e.g., oxygen) can be administered to the microtube (e.g., afterimplantation in the subject) to thereby increase the anti-ischemiceffect of the angiogenic factor.

Targeted delivery of a drug to a tissue-of-interest can be also effectedusing a microtube in which the shell enables diffusion of the drugmolecule therethrough and accordingly, the drug molecule can be releasedthrough the shell pores and/or the distal opening of the microtube atthe desired tissue.

The invention further envisages the use of the microtube of theinvention, which include a molecule-of-interest attached thereto, forguiding cell growth ex vivo or in vivo. For example, neuronal cells canbe placed near or in direct contact with the microtube which is attachedto necessary growth factors and nutrients needed for neuronal growth. Itwill be appreciated that once an initial neuronal growth has occurred exvivo, such a system (i.e., the microtube and the neuronal cells) can beimplanted in a subject in need thereof (e.g., a subject withdegenerated, damaged or injured neuronal cells) to thereby enableneuronal growth and guidance.

The microtube of some embodiments of the invention can be included in akit/article of manufacture along with a packaging material and/orinstructions for use in any of the above described methods orapplications.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”. This termencompasses the terms “consisting of” and “consisting essentially of”.

The phrase “consisting essentially of” means that the composition ormethod may include additional ingredients and/or steps, but only if theadditional ingredients and/or steps do not materially alter the basicand novel characteristics of the claimed composition or method.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

An electrospinning apparatus can include a controller programmed withparameters as described herein or measuring output and automaticallymodifying. Controller can be hardware, software, firmware, with CPU,volatile memory, optional non-volatile memory.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate some embodiments of the invention in anon limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

General Materials and Experimental Methods

Enzymes and Solutions—

The compositions of the shell and core solutions are given in Table 3,hereinbelow. All polymers and solvents were purchased from Sigma-Aldrichand were used as is. Alkaline phosphatase (AP) and beta-galactosidase((3-GAL) from E. coli were also purchased from Sigma-Aldrich. AP cleavesmonophosphate esters and has a molecular weight of about 80,000 Da.β-GAL is a tetrameric enzyme of 465,396 Da consisting of four identicalsubunits each (Zabin I., et al., 1980) and catalyzes the hydrolysis ofthe terminal galactosidyl group of β-galactosides. Both enzymes wereinitially dissolved in water and then mixed with the core solution.

TABLE 3 Two types of core-shell microtubes: composition of the solutionsType Shell solution Core solution 1 10% PCL 80 K; in 4% (w/w) PEO 600K + 0.0733 mg/ml CHCl₃:DMF (90:10 by AP or 2.38 units/ml β-GAL; inweight) ethanol:H₂O (26:74 by weight) 2 10% PCL 80 K + 1% 4% (w/w) PEO600 K + 0.0733 mg/ml PEG 6 K; in AP or 2.38 units/ml β-GAL; in CHCl₃:DMF(90:10 ethanol:H₂O (26:74 by weight) by weight) Table 3. Microtubes wereformed by co-electrospinning of the shell solution (a first polymericsolution for forming the shell) and a core solution (a second polymericsolution for forming the coat over the internal surface of the shell).

Electrospinning—

Hollow microtubes (core-shell hollow fibers) were fabricated by aco-electrospinning process using the set up described by Sun et al. 2003and Zussman et al. 2006. All experiments were conducted at roomtemperature (about 22° C.) and a relative humidity of about 35%. Thespinning parameters were as follow: the electrostatic field used wasapproximately 0.44 kV/cm and the distance between the spinneret andcollector plate was 16 cm. The flow rates of both the core and shellsolutions were controlled by two syringe pumps and were 3.5 ml/hour forthe shell solution and 1 ml/hour for the core solution. The fibers werecollected as a strip on the edge of a vertical rotating wheel (TheronA., et al., 2001) having a velocity of 1.2 m/second. For fluorescencemicroscopy, a few fibers were collected directly onto a microscope slide

Imaging—

Images of the fibers were obtained using a Leo Gemini high resolutionscanning electron microscope (HRSEM) at an acceleration voltage of 3 kVand a sample to detector distance of 3-5 mm. The specimens were coatedwith a thin gold film to increase their conductivity. Fluorescencemicroscope Leica D M IRE2 at excitation and emission wave lengths of 359and 361 nm, respectively, was used for the imaging of fibers filled withfluorescent product.

Measurement of the Enzyme Activity—

To measure enzyme activity, pieces of mat were weighed and dipped inassay solution according to Table 4, hereinbelow. At each time ofsampling, the solution was mixed with a vortex mixer, and 1 ml of theassay mixture was transferred to spectrophotometer cuvette. Theabsorbance of the solution was measured in a Perkins-Elmerspectrophotometer at a wavelength of 410 nm. For both enzymaticreactions, the substrates are colorless but the products,para-nitrophenol for AP and ortho-nitrophenol for β-GAL, are yellow withan absorption maximum at 410 nm. After the absorbance was measured, theliquid was returned to the assay vessel. Units, activity and relativeactivity are defined as follow:

$\begin{matrix}{{Unit} = {\frac{\Delta \; A}{\Delta \; t} \cdot 1000}} & (1) \\{{{{Activity}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {mat}} = {{unit} \cdot C}}{where}{C = \frac{{mass}\mspace{14mu} {of}\mspace{14mu} {total}\mspace{14mu} {mat}}{{mass}\mspace{14mu} {of}\mspace{14mu} {piece}}}} & (2) \\{{{Relative}\mspace{14mu} {{Activity}(\%)}} = \frac{{Activity}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {mat}}{{Activity}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {core}\mspace{14mu} {solution}}} & (3)\end{matrix}$

Where: ΔA is the difference is the absorbance at difference times, Dt,difference between t₁ and t₂ (two time points) taken in the linearregion of the reaction curve, and C is a normalization factor whichtakes into account the different weight of each piece.

For the fluorescence microscope imaging a drop of the assay solution wasput directly on the microscope slide on which a few fibers had beendeposited. The fluorescent substrates were methylumbelliferyl-phosphatefor alkaline phosphatase (AP) and methylumbelliferyl-galactoside forbeta-galactosidase (β-GAL). These were used at the same concentrationsas their nitro-phenyl analogs.

TABLE 4 Composition of the assays Enzyme Substrate Buffer H₂O ProductsAP 0.7 mg/ml p-nitrophenyl- TRIS- 0.5 ml p-nitrophenol + PO₄ phosphate(MW = 217 HCl Da) buffer 1 ml 1.5 ml 4-methylumbelliferyl-4-methylumbelliferone + PO₄ phosphate: fluorescence microscopy β-GAL 4mg/ml o-nitrophenyl-β- Z-buffer 0.3 ml o-nitrophenol + D-galactoside 0.7ml galactose 0.2 ml (MW = 301 Da) — 4-methylumbelliferyl-β-4-methylumbelliferone + galactose D-galactoside: fluorescence microscopyTable 4.

Example 1 Attachment of Enzymes to Electrospun Microtubes

Experimental Results

Formation of Micro-Tubes—

Two types of electrospun hollow fibers have been fabricated with thepolymers listed in Table 3, hereinabove. The resultant fibers are hollowstructures, namely micro-tubes, as previously described by the presentinventors (Dror, Y et al., 2007 and PCT/IB2007/054001, which is fullyincorporated herein by reference). The hollow nature of these structuresand the different morphologies of the tube walls are demonstrated in thehigh resolution scanning electron microscope (HRSEM) micrographspresented in FIGS. 1a-d . Type 1 fibers are made with only PCL in theshell and exhibit a rough surface due to the rapid evaporation of thesolvent (FIGS. 1a and c ). However, this roughness doesn't affect theintact nature of the walls. As PEG is added to the shell solution (Type2), the walls become increasingly porous (FIGS. 1b and d ) and pores canbe seen even in the interior surface of the tubes (FIG. 1 b). PEG andPCL are partially miscible due to favorable, but weak, intermolecularpolar-interactions (Coleman M M, et al., 1991). During fibersolidification along with the evaporation of the solvents, theconcentration of the components increases and phase separation takesplace. However, since the PEG has a surfactant-like character itdeposits an adherent film around the PCL domains resulting in theformation of pores rather than forming solid domains of PEG.

After electrospinning the tubes form a fibrous non-woven, aligned ornon-aligned mat. When the mat material is placed in an aqueousenvironment, the coat over the internal surface of the shell dissolvesand the enzymes are released (through desorption) and become active ontheir substrate(s). For a further description of the desorption processsee FIG. 8 and Example 8 hereinbelow. This arrangement of materialallows for flow-through technologies without subjecting the enzyme tothe external environment and without the need for chemical attachmentand can prevent loss of the entrapped enzyme.

Enzymes Attached to Fibers Maintain Normal Biological Activity—

The kinetics of the enzymatic reaction for alkaline phosphatase wasmeasured as described in the experimental section and is presented inFIGS. 2a-b . The enzymatic reaction with the fibers was compared to thefree enzyme in the core solution (before electrospinning) and normalizedwith respect to the weight of the analyzed pieces. The results stronglyindicate that the enzyme attached in the fiber (e.g., encapsulated)maintains its biological activity after electrospinning and exhibits areaction curve similar to the free enzyme. The curves are characteristicof enzymatic reactions when there is large excess of substrate. Thecolored product, p-nitrophenol, diffuses out of the fibers into thesurrounding medium as shown in FIG. 2c (Type 1). The reaction rates ofthe enzyme attached (e.g., contained within) the microtubes are slightlyreduced in comparison to the free enzyme. Without being bound by anytheory, it is possible that the reaction rates are reduced because ofthe following reasons: (1) the substrate has to diffuse into the fibersin order to reach the active site of the enzyme; and/or (2) the enzymehas to diffuse outside; (3) the product has to diffuse outside in orderto be detected; (4) some enzyme activity was lost during the spinningprocess itself. The attainment of maximum reaction velocity occurs morerapidly with Type 2 fibers, undoubtedly due to their highly porouscharacter. Upon closer examination of the initial kinetics, it can beseen that Type 1 fibers, which do not contain PEG in the shell, exhibita linear reaction rate that is much reduced (inset—FIG. 2b , marked bydark arrows) than Type 2 fibers (which include PEG in the shell polymer,and consequently, pores in the microtube shell). Without being bound byany theory, the initial low rate seems to result from the time requiredfor the penetration of the substrate into the fibers since these fibershave a less porous morphology and are more hydrophobic. In the porousfibers (Type 2) the penetration of the substrate is barely hamperedsince the presence of PEG facilitates the wetting of the outer surfaceof the fibers and thereby allows the access of the aqueous substrate.

Altogether, these results demonstrate that enzymes attached to (e.g.,encapsulated within) electrospun microtubes maintain their enzymaticactivity.

Thus, the present technology overcomes both the problem of fiberdissolution and subsequent leaching of the enzyme and the exposure ofthe enzyme to harmful solvents.

Example 2 Electrospun Microtubes can Release Enzymes Attached Thereto

Experimental Results

Enzyme can be Leached Out of the Electrospun Microtubes—

In order to determined whether the substrate diffuses into the fibers orthe enzyme diffuses out of the fibers, 3 specimens containing alkalinephosphatase (AP) were cut out from the non-woven mat and placed inbuffer. The buffer from the first sample was taken immediately andassayed (initial rinse). For the second and third samples the buffer wastaken and assayed after 24 hours and 72 hours, respectively. The firstsample was tested in order to evaluate if any removable enzyme resideson the outer surface of the fibers after the spinning. The relativeactivity of the escaped enzyme in the buffers for the two types offibers is presented in FIG. 3. The results clearly indicate that asignificant fraction of the enzyme has leached out of the fibers withinthe first 24 hours. Hence it can be concluded that the reactionmonitored for the mat (the electrospun microtubes) is a result of bothin-fiber and out-fiber reactions. Interestingly however, for Type 1fibers, which do not contain PEG in the shell, 82% of enzyme hasdiffused outside the fibers within 24 hours (compared to the mat). ForType 2 fibers, which are more hydrophilic due to PEG, the leaching ofthe enzyme is less although by no means negligible. The results alsopoint out, as was already mentioned, that Type 2 fibers are the mostactive system and attains 55% of the activity of the free enzyme.

Alkaline Phosphatase can Migrate to the Outer Surface of the MicrotubesDuring Electrospinning—

Moreover, it is clear that some enzyme is located on the outer surfaceof the fibers (i.e., attached to the microtube shell) and canimmediately enter the surrounding buffer upon rinsing. The migration ofthe enzyme to the outer surface of the fibers during the spinning ismore pronounced in Type 2 and is probably due to PEG which might serveas hydrophilic conduit for the enzyme. It has been already found (ReznikS N, et al., 2006) that in the core-shell process all chargesimmediately accumulate at the outer surface of the shell in the drop.Ions are preferentially subjected to this migration as the electricfield is applied. In the present case, the protein, which is a chargedmolecule, can also migrate to the outer surface during electrospinning.This may explain the relatively large quantity of enzyme that isreleased by rinsing.

Altogether, these results demonstrate that type 2 fibers attain moreenzymatic activity as compared to type 1 fibers. In addition, the enzymewithin the type 2 fibers (which include PEG in the shell) is present inboth the outer surface and the inner surface of the microtubes. Thus,these results demonstrate that the even though the enzyme is mixed withthe core solution, it is capable of migrating into the outer surface ofthe shell during the electrospinning process.

Example 3 Electrospun Microtubes can Serve as Microreactors

Experimental Results

β-Galactosidase Remains within the Hollow Fiber Micro-Reactor—

The kinetics of the β-GAL reaction were determined as describedhereinabove for AP and the results are presented in FIGS. 4a-b .Briefly, pieces of mat (eletrospun microtubes) were immersed in bufferfor a quick rinse, 24 or 72 hours to determine if there is any leachingof the enzyme (FIG. 5, shown are results of Type 2 as an example). Twostriking differences between the AP and β-GAL series were observed (FIG.6): (i) the β-GAL reaction rate of the Type 1 mat is much slowerrelative to that found for AP, and the activity of AP in the type 1 matis slower relative to that found for the activity of the free enzyme inthe core solution (prior to electrospinning). In type 2, conversely, theactivity of the β-GAL is higher than that of AP and the reactionvelocity of the β-GAL enzyme is comparable to the free enzyme. Thisintense response of Type 2 is attributed to the high porosity of thesefibers and their hydrophilic nature; (ii) the results shown in FIG. 5for Type 2 fibers demonstrate that the β-GAL enzyme doesn't diffuse outof the fibers even after 72 hours even though Type 2 might be thought tobe the more likely to allow the enzyme to escape. That is, the reactiontakes place only within the fibers. Thus, for β-GAL, the hollow fibersact as a micro-reactor; the substrate which enters the “reactor” throughthe entire porous shell is cleaved by the encapsulated enzyme and thereaction product then diffuses out into the surrounding medium. Thus,without being bound by any theory it seems that the reduction in thereaction rate for Type 1 is related solely to the slow diffusion of thesubstrate into the fibers. The size of the enzyme seems to affect boththe amount of enzyme that can migrate during the spinning to the outersurface of the fibers and its subsequent escape into the surroundingmedium. In the case of β-GAL only a small amount of enzyme was detectedin the rinsing buffer for Type 2 while for Type 1 no enzyme was detectedat all (data not shown). As was argued before, in the fibers which donot contain PEG in the shell (Type 1) an additional moderate slope atthe very beginning of the reaction can be observed (FIG. 4b -inset,marked by dark arrow) due to the relatively high hydrophobicity of thesurface which hinders the access of the aqueous dissolved substrate.

Altogether, these results demonstrate that the Type 1 system results inlowered enzymatic activity, especially for large proteins such as β-GAL,which cannot diffuse through the shell pores. The fact that Type 1fibers are hydrophobic and non-porous and thus inhibit the entrance ofthese substrates seems to make Type 1 fibers less efficacious for theiruse as flow-through reactors. This is in contrast to the remarkablyefficient system obtained with Type 2 fibers.

The Enzymatic Reaction Occurs within Type 1 Microtubes (Hollow Fibers)—

Visual evidence that the enzymatic reaction occurs within the type 1fibers was obtained by using a substrate in which one of the products isfluorescent. For both AP and β-GAL enzymes the substrates used liberate4-methyumbelliferone after hydrolysis which allows imaging byfluorescence microscopy. As is clearly seen in FIGS. 7a-b , the interiorof the fibers is fluorescent while the surrounding medium is dark. Thus,these results clearly show that both enzymatic reactions (of AP andβ-GAL), in fact, take place within the fibers. It is important toemphasize that, in contrast to the mat immersion experiments, thismethod is very sensitive and enables the detection of very small amountsof product which accumulates within a relatively short time. Indeed,these images were acquired within 1-2 minutes after the substrate wasapplied, a time scale which is larger than the characteristic time[about 10 seconds, as was calculated by the present inventors (Dror Y.,et al., 2007)] of the diffusion through the micro-tubes wall. Hence,these results are not in contradiction to those of the mat immersionexperiments in which the kinetics were followed over a much longerperiod during which both the product and the enzyme (in the AP case) candiffuse outside the fibers. In FIG. 7b short slugs (sections) offluorescent liquid are observed. This phenomenon has been previouslyfound in such fibers (Dror Y., et al., 2007).

The results shown in Examples 1-3 demonstrate the direct incorporationof enzymes into micro-tubes fabricated by co-electrospinning byintroduction of the enzymes into the aqueous core solution of themicrotube (e.g., PEO). The shell solution in this case was made of PCLdissolved in mixture of chloroform and DMF. The separation between theouter organic and inner aqueous phases was found to preserve enzymeactivity during and after spinning when the electrospun fibers (mats)were subsequently placed in an aqueous environment.

Two types of micro-tubes were fabricated which differ in their shellmorphology. Type 2 shells were produced by adding PEG to the shellsolution. By using a mixture of PEG and PCL in the shell, pores wereformed during the solidification process and this, in turn, directlyaffected the transfer of molecules into and out of the fibers. As aconsequence, the more porous fibers (Type 2) exhibited higher rates ofenzymatic reaction. In addition two enzymes differing in their molecularweight were incorporated: AP and β-GAL. The difference in the molecularweight between the enzymes was well reflected in the kinetics of theenzymatic reactions for both types of micro-tubes. While AP coulddiffuse outside the fibers, β-GAL remained in the fibers without anyleaching of the enzyme and the progress of the reaction depended only onthe arrival of the substrate from the surroundings. Thus, the AP fibersact as an enzyme release device in which the release rate can betailored by modifying the morphology of the shell and, on the otherhand, the β-GAL fibers act as an enzymatic micro-reactor with anefficient provision of the substrate through the entire surface area andefficient discharge of the product. Thus, by manipulating the morphologyof the shell, the substrate supply and product release rate can becontrolled. This method of encapsulation can be used when a separationbetween the enzyme and an external aqueous environment is desired (e.g.with living tissue to avoid immunological reactions). The remarkableretention of the enzyme activity for β-GAL Type 2 fibers clearlydemonstrates that this approach preserves the activity of the enzyme.

Another advantage of the core-shell fiber method is the small volumewithin the micro-tubes which enables the quick buildup of the product.This is important for enzymes working in sequence where the localconcentration of the product of the first reaction serves as thesubstrate for a subsequent reaction. In this regard, these nanotubes aresomewhat analogous to living cells except that any manner of enzymes maybe added to the fibers without regard to their biological origin.Another advantage of this system is that unlike living cells, there isno discrimination as to which type of small molecules may enter thesetubes. For example, phosphorylated molecules (like p-nitrophenylphosphate) which do not enter Escherichia coli cells can enter themicrotubes described herein.

Example 4 Use of the Electrospun Microtubes as Microreactors forChemical Transitions

The Microtubes of the Invention as Reactions for Multi-Step EnzymaticProcesses—

In order to synthesize or catabolize molecules which require multi-stepenzymatic processes the present inventors have devised electrospunmicrotubes which include the enzymes participating in the multi-stepenzymatic process, attached thereto, as follows.

For a biochemical pathway which involves the conversion of A to E viacompounds B, C and D, the second polymeric solution for forming the coatover the internal surface of the shell (also referred to as a coresolution) is mixed with the following enzymes: the enzyme that convertsthe starting substrate A to intermediate compound B, the enzyme thatconverts B to C, the enzyme that converts C to D, and the enzyme thatconverts D to the end product E (see for example, FIG. 9). It should benoted that due to the proximity of the enzymes to each other in themicrotube (which can be a closed micro-reactor), the localconcentrations of each of the intermediate molecules, i.e., compounds B,C, and D is relatively high, which enables the kinetics of the reactionsto occur, similarly to their concentrations in cells or cellcompartments (e.g., mitochondria). The shell solution is made fromhydrophobic polymers (water insoluble polymers), with or without theaddition of PEG.

Thus, the microtube of the invention enables higher local concentrationsof intermediate compounds which can not be reached from the samestarting material (substrate A) if an open system (such as any solidsubstrate to which an enzyme is immobilized) is used.

The creation of micro- and nano-fibers containing enzymes simulates thecellular structure because two or more different enzymes involved in aparticular synthesis or degradation can be put into proximity of oneanother. The interior of the tube is quite parallel to that of cellsexcept that the borders of the tube are made from a water insolublesubstance whereas living cells are encompassed by lipid membranes. Inaddition the microtubes are much longer than cells but quite similar inother dimensions to a bacterial cell. In the electrospun fibers, anysmall molecule can pass through the water insoluble barrier (pores canbe made) regardless of its chemistry with the only provision that thesmall molecule be water soluble.

Thus, the present technology allows the entrapment of highconcentrations of an enzyme or several enzymes within a confined space.Single or multi-step reactions can then take place where the product ofone reaction is the substrate of a second and the second enzyme isspatially nearby. While such multi-step reactions can occur in an opensystem, the time necessary to reach the end product is orders ofmagnitude greater than within the microtube of the invention.

The Microtubes of the Invention can Include Enzymes from DifferentSpecies—

Another very important advantage of these electrospun fibers is thatthere is no limitation of which enzymes can be embedded. In nature,cells contain enzymes useful for their growth and reproduction.Organisms have not been designed or selected for industrial processesdesired by humans. The microtubes of the invention allow any desiredcombination of enzymes to be brought together. This might mean thatenzymes from totally different organisms (e.g. flies and humans) couldbe placed together for some use while in nature no organism exists withthis combination.

The Microtubes of the Invention as Micro-Reactors for the Production ofMolecules which are Intermediate Compounds of a Natural Process—

The enzymes encapsulated might be those carrying out part of pathwaymaking the end product a substance that is usually an intermediatemolecule in living organisms. This allows one to synthesize moleculesthat cannot be obtained in any amount from living material because theconcentration of intermediates in cells is usually very low (in theorder of 10 μM or less). For example, to synthesize indole-glycerolphosphate which is an intermediate in tryptophan synthesis within thecells of lower organisms, the enzymes that participate in the conversionof anthranilate (an inexpensive compound) to indole-glycerol phosphateshould be included in the microtube, while the enzymes that continue thesynthesis of tryptophan from indole-glycerol phosphate are excluded fromthe microtube. In summary, many combinations of enzymes from differentorganisms may be put together without any genetic engineering andpartial sets of enzymes can also be used. The number of possible usefulcombinations is therefore very large.

Thus, the microtubes of the invention can be used as enzymaticmicro-reactors where the inner space enables a confined but freereaction space. The substrate diffuses through the shell to the innerspace where the enzymatic reaction takes place and the product can thendiffuse out.

Example 5 The Electrospun Microtubes as Biosensors

Since the electrospun microtubes of the invention are insoluble inaqueous solutions, they can provide an excellent tool for theconstruction of biosensors.

Since any enzyme or combination of enzymes can be encapsulated in theelectrospun microtubes, a variety of biosensors can be devised. Forexample, enzymes that are sensitive to heavy metals exhibit loss ofactivity in the presence of heavy metals. Another example, fireflyluciferase, for example can be electrospun with its luciferin cofactorand any reaction affecting ATP production can be used in conjunctionwith light output the signal.

Example 6 The Electrospun Microtubes for Flow-Through Applications

Water Purification or Detoxification—

The electrospun microtubes of the invention, which are made of awater-insoluble outer shell, enable the flow of liquids. It will beappreciated that enzymes embedded in such microtubes can be used topurify the liquid flowing past the microtubes as molecules diffuse inand out of them.

Thus the present inventors have devised water purification ordetoxification apparatuses, as follows. The second polymeric solutionwhich forms the coat over the internal surface of the shell (alsoreferred to as a core solution) [which is made of water-solublepolymer(s)] includes enzymes which remove a toxic moiety from water,such as the gene product of the atzA gene from Pseudomonas ADP thatremoves the chlorine from atrazine, a toxic substance. The shellsolution [which is made of water-insoluble polymer(s)] is designed so asto enable water flow within the microtube. The effluent would thereby berendered free of atrazine and safe for animal and human consumption.

Dialysis—

The microtubes of the invention can be used in various applicationswhich remove certain compounds, such as dialysis procedures on humans.Thus, the electrospun microtubes can be made using a shell polymer whichprevents the diffusion of enzymes therethrough, yet enables passage ofwater and substances that need to be purified. It will be appreciatedthat such microtubes can be also implanted into a subject in needthereof (e.g., a subject in need of dialysis), and due to the structureof a closed conduit, which prevents passage of embedded enzymes throughthe shell, there is no immune response to the implanted conduit.

Example 7 The Electrospun Microtubes for Enzyme Therapy

Since the electrospun microtubes of the invention are insoluble inaqueous solutions, they should provide excellent tool for theconstruction of material for enzyme therapy. Some individuals lackcertain enzymes, usually as a result of their being homozygous forrecessive alleles that lead to synthesis of an inactive enzyme. Genetherapy attempts to introduce the missing active gene which therebyleads to the production of an active enzyme. However, this technique isstill quite inefficient. A different way of treating such patients isenzyme therapy, in which the missing enzyme is exogenously supplied tothe subject. The main drawbacks of the second method is that injectionof enzymes often leads to the formation of antibodies against them andthe half-life of the enzymes within the body may be quite short.

Thus, the present inventors have devised an apparatus for enzymetherapy, as follows. Briefly, the electrospun microtubes which include awater-insoluble shell can include enzymes which are needed for enzymetherapy, and be further implanted in a subject in need thereof.

Enzyme Therapy for PKU—

Phenylketonuria (PKU) occurs in slightly less than 1 per 10000individuals and is an autosomal recessive genetic disease caused byhomozygosity of alleles encoding defective enzymes. Phenylalanine andtyrosine are amino acids that are found in most proteins. In humans, thesource of these two amino acids is dietary protein. In normalindividuals, excess phenylalanine is converted to tyrosine. Excesstyrosine, in turn, is broken down to fumarate and acetoacetate. Bothtyrosine and phenylalanine are essential for human protein synthesis. Inaddition, tyrosine is the precursor of melanin (skin and eye pigment)and for certain hormone like substances such as thyroxine.Phenylketonuria is caused by the lack of the enzyme (phenylalanine4-monooxygease EC 1.14.16.1) that converts phenylalanine to tyrosine.The result of this defect is the accumulation of phenylalanine in theblood along with a number of compounds that are derived from it (e.g.phenylpyruvic acid and phenyllactic acid). The result is brain damage(and an IQ of 30-70) as some of these compounds are toxic. The currentmethod of preventing deterioration of the disease is to limit the intakeof phenylalanine. The present inventors have envisaged that PKU can betreated by implanting electrospun microtubes containing the missingenzyme, phenylalanine hydroxylase (PAH; GenBank Accession No. NP_00026),in a subject diagnosed with PKU, and thereby enabling the breakdown ofexcess phenylalanine in the subject. It will be appreciated that in thiscase, the microtube can be designed so as to enable release of enzymefrom the inner surface through the outer shell (e.g., using PEG in theouter shell) or alternatively can be designed such that the enzyme isentrapped (or remains) within the microtubes and effects its activitythere (e.g., by diffusion of the substrate or end-product through theshell pores, or microtube opening(s)). As phenylalanine hydroxylase canbe phosphorylated (with a molecular weight of consists of 50,000 Da) ordephosphorylated (with a molecular weight of 49,000 Da), the size of thepores in the shell should enable passage (by diffusion) of each of theseforms (e.g., about 5 nm in diameter). Microtubes can be made withsmaller pores that will prevent the loss of the enzyme which will remainwithin the microtube.

Example 8 The Desorption Process

FIG. 8 schematically depicts the desorption of a molecule-of-interest tothe microtube of the invention. After the electrospinning process, themolecule-of-interest (e.g., a protein, an enzyme) is adsorbed to theinner side of the microtubes. As mentioned, the porosity of themicrotubes can be controlled (e.g., adding PEG to the shell polymer),therefore the shell consists of nanopores (see #3 in FIG. 8) with anopening to the outer surface of the microtube, which herein the poresare considered to have a cylindrical shape. When immersing themicrotubes in a solution (e.g., a tissue culture medium, a physiologicalsolution or any buffer) most nanopores opening are accessible to thesolution.

Once the microtubes are immersed in the solution the nanopores arefilled by the solution through capillary rise (see arrow #1 in FIG. 8).It will be appreciated that the time of the capillary rise depends onthe solution rheological properties (viscosity and surface tension), thewetting angle and the geometry of the nanopore (length and radius). Thesolution penetrates to the microtube and start wetting and filling itsentire inner volume. Desorption of the molecule-of-interest from themicrotube wall depends mainly on the rate of the release of themolecule-of-interest from the polymer of the second polymeric solution.Finally, the molecule-of-interest (e.g., protein/enzymes) diffuses (seearrow #2 in FIG. 8) into the solution and released to the surroundingsof the microtubes (assuming that the major release is through themicrotubes envelope.

Note that the geometry (radius and length) of the nanopore iscontrollable, by adjusting the shell thickness, or by blending, more PEGto the shell polymer. Therefore, the release from or confinement in themicrotube of the invention is controllable, e.g., in certain cases themolecule-of-interest is released from the microtube, whereas in othercases, the molecule-of-interest remains in the inner volume of themicrotube. As shown in FIG. 2a , a molecule-of-interest is released fromthe microtube of the invention in a controlled manner, which can beextended beyond 2500, minutes, e.g., for several days and months.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

REFERENCES Additional References are Cited in Text

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1. A microtube comprising: an electrospun shell, an electrospun coatpolymer over an internal surface of said shell and amolecule-of-interest attached to the microtube, wherein said electrospunshell is formed of a first polymeric solution comprising a first solventand said electrospun coat is formed of a second polymeric solutioncomprising a second solvent, wherein said second solvent of said secondpolymeric solution is incapable of dissolving a polymer of said firstpolymeric solution, wherein said first polymeric solution solidifiesfaster than said second polymeric solution, wherein said secondpolymeric solution is capable of wetting said internal surface of saidshell during or following solidification of said first polymericsolution, wherein said molecule-of-interest is selected from the groupconsisting of: a polypeptide, a polynucleotide, a carbohydrate, apolysaccharide, a lipid, a drug molecule, and a small molecule, andwherein said small molecule is selected from the group consisting of anucleotide base, an amino acid, a nucleotide, an antibiotic, and avitamin.
 2. The microtube of claim 1, wherein said polymer of said firstpolymeric solution and a polymer of said second polymeric solution aredifferent.
 3. The microtube of claim 1, wherein said electrospun shellcomprises pores.
 4. The microtube of claim 1, wherein said electrospunshell comprises a polymer selected from the group consisting of poly(e-caprolactone) (PCL), polyamide, poly(siloxane), poly(silicone),poly(ethylene), poly(vinyl pyrrolidone), poly(2-hydroxyethylmethacrylate), poly(N-vinyl pyrrolidone), poly(methylmethacrylate), poly(vinyl alcohol), poly(acrylic acid), poly(vinylacetate), polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethyleneglycol), poly(methacrylic acid), polylactide, polyglycolide,poly(lactide-coglycolide), polyanhydride, polyorthoester,poly(carbonate), poly(acrylo nitrile), poly(ethylene oxide),polyaniline, polyvinyl carbazole, polystyrene, poly(vinyl phenol),polyhydroxy acid, poly(caprolactone), polyanhydride,polyhydroxyalkanoate, polyurethane, collagen, albumin, alginate,chitosan, starch, and hyaluronic acid.
 5. The microtube of claim 1,wherein said electrospun coat comprises a polymer selected from thegroup consisting of poly(acrylic acid), poly(vinyl acetate),polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethylene glycol),poly(methacrylic acid), polylactide polyglycolide,poly(lactide-coglycolide), polyanhydride, polyorthoester,poly(carbonate), poly(ethylene oxide), polyaniline, polyvinyl carbazole,polystyrene, poly(vinyl phenol), polyhydroxy acid, alginate, starch,hyaluronic acid.
 6. The microtube of claim 1, wherein at least one ofelectrospun shell and electrospun coat comprises a polymer selected fromthe group consisting of: collagen, albumin, alginate, chitosan, starch,and hyaluronic acid. elastin, tropoelastin, thrombin, fibronectin,poly(amino acids), poly(propylene fumarate), gelatin, pectin, fibrin,cellulose, oxidized cellulose, chitin, polyethylene, polyethyleneterephthalate, poly(tetrafluoroethylene), polycarbonate, andpolypropylene, or derivatives thereof.
 7. The microtube of claim 1,wherein said first solvent of said first polymeric solution evaporatesfaster than said second solvent of said second polymeric solution, andwherein said second solvent of said second polymeric solution is capableof evaporating through said internal surface of said shell.
 8. Themicrotube of claim 1, wherein a thickness of said shell is from about100 nm to about 20 micrometer.
 9. The microtube of claim 1, wherein aninternal diameter of the microtube is from about 50 nm to about 20micrometer.
 10. The microtube of claim 1, wherein said microtube isfilled with a liquid.
 11. The microtube of claim 1, wherein saidmolecule-of-interest is attached to said coat over said internal surfaceof said shell.
 12. The microtube of claim 1, wherein saidmolecule-of-interest is attached to said shell of the microtube.
 13. Themicrotube of claim 1, wherein said first polymeric solution furthercomprises polyethylene glycol (PEG).
 14. The microtube of claim 1,wherein said shell prevents diffusion of the molecule-of-interesttherethrough.
 15. A microfluidic device comprising a plurality of themicrotubes of claim
 1. 16. The microtube of claim 1, wherein at leastone of said first polymeric solution and second polymeric solutioncomprises a co-polymer.
 17. The microtube of claim 1, wherein at leastone of said first polymeric solution and second polymeric solutioncomprises a blend of polymers.
 18. A method of attaching amolecule-of-interest to a microtube, the method comprising:co-electrospinning two polymeric solutions through co-axial capillaries,wherein a first polymeric solution of said two polymeric solutions isfor forming a shell of the microtube and a second polymeric solution ofsaid two polymeric solutions is for forming a coat over an internalsurface of said shell, said first polymeric solution is selectedsolidifying faster than said second polymeric solution and a solvent ofsaid second polymeric solution is selected incapable of dissolving saidfirst polymeric solution and wherein said second polymeric solutioncomprises the molecule-of-interest, thereby attaching themolecule-of-interest to the microtube.
 19. A method of processing asubstrate-of-interest, comprising contacting the substrate-of-interestwith the microtube of claim 1, wherein said molecule-of-interest iscapable of processing said substrate, thereby processing thesubstrate-of-interest.
 20. The method of claim 19, wherein saidprocessing is selected from isolating a molecule and detecting apresence of a molecule in a sample.